Pharmaceutical formulations and methods of use thereof

ABSTRACT

This disclosure is directed to antibody-drug conjugates. More specifically, this disclosure is directed to compositions comprising (i) antibody-drug conjugates comprising benzodiazepines, and (ii) a small hydrophobic molecule, methods of treatment using the compositions, and methods of formulating the compositions. Furthermore, this disclosure is directed to methods of reducing reversible self-association in antibodies and in antibody-drug conjugates.

PRIORITY CLAIM

This application is a continuation application of co-pending U.S.application Ser. No. 15/360,689, filed Nov. 23, 2016, which claimspriority to U.S. Provisional Application No. 62/368,156, filed Jul. 28,2016, and to U.S. Provisional Application No. 62/260,104, filed Nov. 25,2015. The entire disclosures of those applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 14, 2016, isnamed P155_US003_SL.txt and is 22,322 bytes in size.

FIELD

The present disclosure relates to pharmaceuticals. More specifically,this disclosure relates to formulations for antibody-drug conjugates andmethods related to antibody-drug conjugate compositions.

BACKGROUND

In recent years, the treatment of cancer has become more targetedthrough the development of antibody-drug conjugates (herein referred toas “ADCs,” “conjugates,” or “immunoconjugates”). Researchers haveidentified and taken advantage of cell-surface receptors and antigensselectively expressed by cancer cells to develop drugs based onantibodies that bind tumor-specific or tumor-associated antigens. Thisspecific binding allows for the delivery to the cancer cells ofcytotoxic compounds linked to the antibody. The selectivity afforded byADCs minimizes toxicity to normal cells, thereby enhancing tolerabilityof the drug in the patient.

Despite the tumor selectivity afforded by ADCs, the use of ADCs in aclinical context is limited by a number of factors. Among those factorsis the ability of the ADCs to aggregate and reversibly self-associate.ADCs are able to aggregate to each other through various mechanisms,including covalent bonding and hydrophobic interactions. ADCs are alsoable to reversibly self-associate through weak interactions that createequilibrium between monomers and higher ordered species. In either case,aggregation and reversible self-association inhibit the ability of theADC to bind to the target, thereby reducing the clinical efficacy of theADC. Accordingly, researchers continue to work to discover ways to limitaggregation and reversible self-association of the ADCs and increase theefficacy of ADCs.

SUMMARY

This disclosure is directed to ADC compositions with reduced reversibleself-association, methods of treating cancer using such compositions,methods of formulating such compositions, and methods of reducingreversible self-association. In one aspect, this disclosure provides acomposition comprising (a) an ADC comprising at least onebenzodiazepine; and (b) a small hydrophobic molecule selected from thegroup consisting of betaines and amino acids with hydrophobic sidechains. In some embodiments, the composition has a pH between about 4.0and about 4.5. In some embodiments, the benzodiazepine is anindolinobenzodiazepine. In some embodiments, the benzodiazepine isselected from the group consisting of D1, D1(a), D2, D2(a), DGN462,DGN462(a), D3, D3(a), D4, D4(a), D5, D5(a), D6, and D6(a), which aredescribed below in Table 1. In certain embodiments, the ADC is selectedfrom the group consisting of Ab-sSPDB-D1, Ab-sSPDB-D1(a), Ab-D2,Ab-D2(a), Ab-sSPDB-DGN462, Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a),Ab-sSPDB-D4, Ab-sSPDB-D4(a), Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1,Ab-Ser-D1(a), Ab-Cys-DGN462, Ab-Cys-DGN462(a), Ab-Ser-DGN462,Ab-Ser-DGN462(a), Ab-Cys-D5, Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a),which are described below in Table 2. In some embodiments, thecomposition is an aqueous solution.

In some embodiments, the antibody is selected from the group consistingof huMy9-6, huB4, huDS6, huMov19, and huCD37-3. In other embodiments,the antibody is a humanized CD123 antibody. In certain embodiments, theantibody is a humanized CD123 antibody described in U.S. Provisionalapplication No. 62/186,161, U.S. Patent Application Publication No.US20170029514A1, and PCT Application publication no. WO2017004026, whichare herein incorporated by reference in their entireties. As usedherein, “AbX” refers to humanized CD123 antibodies described in U.S.Provisional Application No. 62/186,161, U.S. Patent ApplicationPublication No. US20170029514A1, and PCT Application publication no.WO2017004026. All applications, patents, and other publicationsreferenced herein are incorporated by reference. In some embodiments,the AbX antibody comprises the CDR sequences disclosed herein. In someembodiments, the AbX antibody comprises the heavy chain variable regiondomain sequences and light chain variable region domain sequencesdisclosed herein.

In further embodiments, the composition is a lyophilized composition. Instill further embodiments, the composition is a reconstitutedlyophilized composition. Although the small hydrophobic molecule can beadded to compositions prior to lyophilization, the benefits of reducedor inhibited reduced reversible self-association are still realized incompositions that are reconstituted after lyophilization.

In some embodiments, the small hydrophobic molecule is trimethylglycine.In certain embodiments, the small hydrophobic molecule is proline. Inother embodiments, the small hydrophobic molecule is leucine. In furtherembodiments, the small hydrophobic molecule is isoleucine. In stillfurther embodiments, two or more small hydrophobic molecules are used incombination in the compositions.

The number of benzodiazepines per antibody in an ADC can vary. In someembodiments, the ADC comprises at least one benzodiazepine. In certainembodiments, the ADC comprises at least two benzodiazepines. In furtherembodiments, the ADC comprises at least three benzodiazepines. In stillfurther embodiments, the ADC comprises at least four benzodiazepines. Inother embodiments, the ADC comprises at least five benzodiazepines. Incertain embodiments, the ADC comprises at least six benzodiazepines. Insome embodiments, the ADC comprises about seven benzodiazepines. Incompositions comprising more than one ADC, the average number ofbenzodiazepines per antibody can be measured. This number is referred toas the drug-to-antibody ratio, or “DAR.” In some embodiments, acomposition comprising more than one ADC has a DAR between about 1 andabout 4. In some embodiments, a composition comprising more than one ADChas a DAR between 0 and about 1. In other embodiments, a compositioncomprising more than one ADC has a DAR between about 1 and about 2. Instill other embodiments, a composition comprising more than one ADC hasa DAR between about 2 and about 3. In further embodiments, a compositioncomprising more than one ADC has a DAR of between about 3 and about 4.In still further embodiments, a composition comprising more than one ADChas a DAR between about 4 and about 5. In yet further embodiments, acomposition comprising more than one ADC has a DAR between about 5 andabout 6. In some embodiments the benzodiazepine is conjugated to theantibody in a site-specific manner, for example, through conjugation toan engineered cysteine or serine residue.

In some embodiments, the composition is an aqueous formulationcomprising: (a) water; (b) huMy9-6-sSPDB-DGN462; (c) 10 mM sodiumsuccinate; and (d) 280 mM betaine, wherein the formulation has a pH ofabout 4.2. In another embodiment, the composition is an aqueousformulation comprising: (a) water; (b) huMy9-6-sSPDB-DGN462; (c) 10 mMsodium succinate; and (d) 280 mM proline, wherein the formulation has apH of about 4.2. In yet another embodiment, the composition is anaqueous formulation comprising: (a) water; (b) AbX-D2; (c) 10 mM sodiumsuccinate; and (d) a small hydrophobic molecule selected from the groupconsisting of 280 mM proline and 280 mM betaine, wherein the formulationhas a pH of about 4.2. In one embodiment, the composition is an aqueousformulation comprising: water; (a) huMov19-sSPDB-D1; (b) 10 mM sodiumsuccinate; and (c) 125 mM leucine, wherein the formulation has a pH ofabout 4.2. In another embodiment, the composition is an aqueousformulation comprising: (a) water; (b) huMov19-sSPDB-D4; (c) 10 mMsodium succinate; and (a) 125 mM isoleucine, wherein the formulation hasa pH of about 4.2. In some embodiments, the composition is a lyophilizedcomposition of any of the aqueous compositions described herein.

In another aspect, the disclosure provides a method of treating cancerin a subject, comprising administering to a subject in need thereof aneffective amount of a composition comprising (i) an ADC comprising abenzodiazepine; and (ii) a small hydrophobic molecule selected from thegroup consisting of betaines and amino acids with hydrophobic sidechains, wherein the ADC is cytotoxic in one or more cells, therebytreating the cancer. In some embodiments, the composition is alyophilized composition. In certain embodiments, the composition is areconstituted lyophilized composition. In some embodiments of themethod, the benzodiazepine is selected from the group consisting of D1,D1(a), D2, D2(a), DGN462, DGN462(a), D3, D3(a), D4, D4(a), D5, D5(a),D6, and D6(a). In certain embodiments, the ADC is selected from thegroup consisting of Ab-sSPDB-D1, Ab-sSPDB-D1(a), Ab-D2, Ab-D2(a),Ab-sSPDB-DGN462, Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a), Ab-sSPDB-D4,Ab-sSPDB-D4(a), Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1, Ab-Ser-D1(a),Ab-Cys-DGN462, Ab-Cys-DGN462(a), Ab-Ser-DGN462, Ab-Ser-DGN462(a),Ab-Cys-D5, Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a). In someembodiments, the antibody (Ab) is selected from the group consisting ofhuMy9-6, huB4, huDS6, huMov19, and huCD37-3. In other embodiments, theantibody is a humanized CD123 antibody. In certain embodiments, theantibody is AbX. In some embodiments, the AbX antibody comprises the CDRsequences disclosed herein. In some embodiments, the AbX antibodycomprises the heavy chain variable region domain sequences and lightchain variable region domain sequences disclosed herein.

In some embodiments of the method, the small hydrophobic molecule istrimethylglycine. In further embodiments of the method, the smallhydrophobic molecule is proline. In some embodiments of the method, thesmall hydrophobic molecule is leucine. In certain embodiments of themethod, the small hydrophobic molecule is isoleucine. In still furtherembodiments of the method, two or more small hydrophobic molecules areused in combination in the compositions.

In some embodiments of the method, the composition used in the methodsis one of the specific aqueous formulations described above. In certainembodiments of the method, the composition is a reconstitutedlyophilized composition derived from one of the aqueous formulationsdisclosed herein.

In yet another aspect, this disclosure provides a method of formulatinga composition, comprising (a) providing an ADC comprising abenzodiazepine in an aqueous solution; and (b) adding to the aqueoussolution a small hydrophobic molecule selected from the group consistingof betaines and amino acids with hydrophobic side chains. In someembodiments, the method further comprises adjusting the pH of theaqueous solution to between about 4.0 and about 4.5. In some embodimentsof the method, the benzodiazepine is selected from the group consistingof D1, D1(a), D2, D2(a), DGN462, DGN462(a), D3, D3(a), D4, D4(a), D5,D5(a), D6, and D6(a). In certain embodiments of the method, the ADC isselected from the group consisting of Ab-sSPDB-D1, Ab-sSPDB-D1(a),Ab-D2, Ab-D2(a), Ab-sSPDB-DGN462, Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a),Ab-sSPDB-D4, Ab-sSPDB-D4(a), Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1,Ab-Ser-D1(a), Ab-Cys-DGN462, Ab-Cys-DGN462(a), Ab-Ser-DGN462,Ab-Ser-DGN462(a), Ab-Cys-D5, Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a).In some embodiments, the antibody is selected from the group consistingof huMy9-6, huB4, huDS6, huMov19, and huCD37-3. In other embodiments,the antibody is a humanized CD123 antibody. In certain embodiments, theantibody is AbX. In some embodiments, the AbX antibody comprises the CDRsequences disclosed herein. In some embodiments, the AbX antibodycomprises the heavy chain variable region domain sequences and lightchain variable region domain sequences disclosed herein.

In some embodiments of the method, the small hydrophobic molecule istrimethylglycine. In other embodiments of the method, the smallhydrophobic molecule is leucine. In some embodiments of the method, thesmall hydrophobic molecule is isoleucine. In certain embodiments of themethod, the small hydrophobic molecule is proline. In still otherembodiments of the method, a combination of small hydrophobic moleculesare added.

In further embodiments of the method, the addition of a smallhydrophobic molecule reduces RSA in the aqueous solution by about 30% toabout 40%. In some embodiments, the addition of a small hydrophobicmolecule reduces RSA in the aqueous solution by about 40% to about 50%.In certain embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 50% to about 60%. Infurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 60% to about 70%. In stillfurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 70% to about 80%. In yetfurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 80% to about 90%. In someembodiments, the addition of a small hydrophobic molecule reduces RSA inthe aqueous solution by about 90% to 100%. In still further embodiments,the addition of a small hydrophobic molecule eliminates RSA in theaqueous solution. In some embodiments, the amount of RSA is measured bymultiangle light scattering. In some further embodiments, the amount ofRSA is measured by dynamic light scattering.

In some embodiments, the method further comprises lyophilizing theaqueous solution, thereby obtaining a lyophilized composition. Incertain embodiments, the method further comprises reconstituting thelyophilized composition, thereby creating a reconstituted lyophilizedcomposition. In further embodiments of the method, the addition of asmall hydrophobic molecule reduces RSA in the reconstituted lyophilizedcomposition by about 30% to about 40%. In some embodiments, the additionof a small hydrophobic molecule reduces RSA in the reconstitutedlyophilized composition by about 40% to about 50%. In certainembodiments, the addition of a small hydrophobic molecule reduces RSA inthe reconstituted lyophilized composition by about 50% to about 60%. Infurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the reconstituted lyophilized composition by about 60% toabout 70%. In still further embodiments, the addition of a smallhydrophobic molecule reduces RSA in the reconstituted lyophilizedcomposition by about 60% to about 70%. In yet further embodiments, theaddition of a small hydrophobic molecule reduces RSA in thereconstituted lyophilized composition by about 70% to about 80%. In someembodiments, the addition of a small hydrophobic molecule reduces RSA inthe reconstituted lyophilized composition by about 80% to about 90%. Incertain embodiments, the addition of a small hydrophobic moleculereduces RSA in the reconstituted lyophilized composition by about 90% to100%. In some embodiments, the addition of a small hydrophobic moleculeeliminates RSA in the reconstituted lyophilized composition.

In still another aspect, this disclosure provides a method of reducingreversible self-association, the method comprising (a) providing an ADCcomprising a benzodiazepine in an aqueous solution, wherein the ADCexhibits reversible self association; and (b) adding to the aqueoussolution a small hydrophobic molecule selected from the group consistingof betaines and amino acids with hydrophobic side chains, wherein thesmall hydrophobic molecule reduces reversible self association. In someembodiments, the small hydrophobic molecule is trimethylglycine. Infurther embodiments, the small hydrophobic molecule is proline. Incertain embodiments, the small hydrophobic molecule is leucine. In stillfurther embodiments, the small hydrophobic molecule is isoleucine.

In certain embodiments, the method further comprises detectingreversible self-association. In further embodiments, the method furthercomprises adjusting the pH of the aqueous solution to between about 4.0and about 4.5.

In some embodiments of the method, the benzodiazepine is selected fromthe group consisting of D1, D1(a), D2, D2(a), DGN462, DGN462(a), D3,D3(a), D4, D4(a), D5, D5(a), D6, and D6(a). In certain embodiments ofthe method, the ADC is selected from the group consisting ofAb-sSPDB-D1, Ab-sSPDB-D1(a), Ab-D2, Ab-D2(a), Ab-sSPDB-DGN462,Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a), Ab-sSPDB-D4, Ab-sSPDB-D4(a),Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1, Ab-Ser-D1(a), Ab-Cys-DGN462,Ab-Cys-DGN462(a), Ab-Ser-DGN462, Ab-Ser-DGN462(a), Ab-Cys-D5,Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a). In some embodiments, theantibody is selected from the group consisting of huMy9-6, huB4, huDS6,huMov19, and huCD37-3. In other embodiments, the antibody is a humanizedCD123 antibody. In certain embodiments, the antibody is AbX and refersto humanized CD123 antibodies described in U.S. Provisional ApplicationNo. 62/186,161, U.S. Patent Application Publication No. US20170029514A1,and PCT Application publication no. WO2017004026. In some embodiments,the AbX antibody comprises the CDR sequences disclosed herein. In someembodiments, the AbX antibody comprises the heavy chain variable regiondomain sequences and light chain variable region domain sequencesdisclosed herein.

In some embodiments of the method, the reversible self-association isreduced by about 30% to about 40%. In further embodiments of the method,the reversible self-association is reduced by about 40% to about 50%. Instill further embodiments of the method, the reversible self-associationis reduced by about 50% to about 60%. In yet further embodiments of themethod, the reversible self-association is reduced by about 60% to about70%. In some embodiments of the method, the reversible self-associationis reduced by about 70% to about 80%. In certain embodiments of themethod, the reversible self-association is reduced by about 80% to about90%. In further embodiments of the method, the reversibleself-association is reduced by about 90% to 100%. In certain embodimentsof the method, the reversible self-association is eliminated.

In some embodiments, the method further comprises lyophilizing theaqueous solution, thereby creating a lyophilized composition. In furtherembodiments, the method further comprises reconstituting the lyophilizedcomposition.

It has been discovered that compositions comprising an ADC exhibitreduced reversible self-association when formulated with a buffer (e.g.,succinate buffer) at a pH ranging from about 4.0 to about 4.5.Accordingly, yet another aspect of this disclosure is directed tocompositions comprising an ADC comprising a benzodiazepine, wherein theADC exhibits reversible self-association, and a buffer, wherein thecomposition has a pH ranging from about 4.0 to about 4.5. In someembodiments, the benzodiazepine is selected from the group consisting ofD1, D1(a), D2, D2(a), DGN462, DGN462(a), D3, D3(a), D4, D4(a), D5,D5(a), D6, and D6(a). In certain embodiments, the ADC is selected fromthe group consisting of Ab-sSPDB-D1, Ab-sSPDB-D1(a), Ab-D2, Ab-D2(a),Ab-sSPDB-DGN462, Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a), Ab-sSPDB-D4,Ab-sSPDB-D4(a), Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1, Ab-Ser-D1(a),Ab-Cys-DGN462, Ab-Cys-DGN462(a), Ab-Ser-DGN462, Ab-Ser-DGN462(a),Ab-Cys-D5, Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a). In someembodiments, the antibody is selected from the group consisting ofhuMy9-6, huB4, huDS6, huMov19, and huCD37-3. In other embodiments, theantibody is a humanized CD123 antibody. In certain embodiments, theantibody is AbX and refers to humanized CD123 antibodies described inU.S. Provisional Application No. 62/186,161, U.S. Patent ApplicationPublication No. US20170029514A1, and PCT Application publication no.WO2017004026. In some embodiments, the AbX antibody comprises the CDRsequences disclosed herein. In some embodiments, the AbX antibodycomprises the heavy chain variable region domain sequences and lightchain variable region domain sequences disclosed herein.

In some embodiments, the composition further comprises a sugar. Incertain embodiments, the sugar is trehalose. In some embodiments, thetrehalose is trehalose dihydrate. In other embodiments, the trehalose istrehalose anhydrous. In other embodiments, the sugar is sucrose. In someembodiments, the buffer is succinate. In some embodiments, thecomposition further comprises sodium bisulfite. In some embodiments, thecomposition is an aqueous formulation. In other embodiments, thecomposition is a lyophilized composition. In some embodiments, thecomposition further comprises a bulking agent. In certain embodiments,the bulking agent is glycine. In other embodiments, the bulking agent ismannitol.

In some embodiments, the aqueous formulation comprises (a) water; (b)huMy9-6-sSPDB DGN462; (c) 10 mM sodium succinate; and (d) 8% trehalose,wherein the formulation has a pH ranging from about 4.0 to about 4.5. Incertain embodiments, the aqueous formulation comprises (a) water; (b)AbX D2 or AbX-D2(a); (c) 10 mM sodium succinate; and (d) 8% trehalose,wherein the formulation has a pH ranging from about 4.0 to about 4.5,and optionally includes 2-200 μM sodium bisulfite. In certainembodiments, the aqueous formulation comprises (a) water; (b) AbX-D5 orAbX-D5(a); (c) 10 mM sodium succinate; and (d) 8% trehalose, wherein theformulation has a pH ranging from about 4.0 to about 4.5 and optionallyincludes 2-200 μM sodium bisulfite. In some embodiments, the aqueousformulation comprises (a) water; (b) 2 mg/mL AbX-D5 or AbX-D5(a); (c) 10mM sodium succinate; (d) 8% trehalose dihydrate; (e) 50 μM sodiumbisulfite; and 0.01% (w/v) polysorbate 20, wherein the formulation has apH of about 4.2. In some embodiments, the aqueous formulation comprises(a) water; (b) huMov19-sSPDB D1 or D1(a); (c) 10 mM sodium succinate;and (d) 8% trehalose, wherein the formulation has a pH ranging fromabout 4.0 to about 4.5. In another embodiment, the aqueous formulationcomprises (a) water; (b) huMov19-sSPDB D2 or D2(a); (c) 10 mM sodiumsuccinate; and (d) 8% trehalose. In further embodiments, the aqueousformulation comprises (a) water; (b) huMov19-sSPDB D4; (c) 10 mM sodiumsuccinate; and (d) 8% trehalose, wherein the formulation has a pHranging from about 4.0 to about 4.5. In some embodiments, thecomposition is a lyophilized composition of any of the aqueouscompositions described herein. In some embodiments, the pH of any of thecompositions described above is 4.2. In some embodiments, the AbXantibody comprises the CDR sequences disclosed herein. In someembodiments, the AbX antibody comprises the heavy chain variable regiondomain sequences and light chain variable region domain sequencesdisclosed herein.

In some embodiments, the composition further comprises a surfactant. Insome embodiments, the surfactant is 0.01% polysorbate 20. In someembodiments, the composition further comprises sodium bisulfite. In someembodiments, the composition comprises 2-200 μM sodium bisulfite. Inother embodiments, the composition further comprises 5-100 μM sodiumbisulfite. In certain embodiments, the composition further comprisesabout 50 μM sodium bisulfite.

In some embodiments, the pH of the composition is about 4.2. In someembodiments, the aqueous formulation comprises (a) water; (b) 2 mg/mLAbX-D5 or AbX-D5(a); (c) 10 mM sodium succinate; (d) 8% trehalosedihydrate; (e) 50 μM sodium bisulfite; and 0.01% (w/v) polysorbate 20,wherein the formulation has a pH of about 4.2.

Another aspect of this disclosure is directed to a method of reducingreversible self association, comprising (a) providing an ADC comprisinga benzodiazepine in an aqueous solution at a first pH, wherein the ADCexhibits reversible self association; and (b) adjusting the pH of theaqueous solution to a second pH ranging from about 4.0 to about 4.5,wherein the adjustment of the pH from the first pH to the second pHreduces reversible self association. In some embodiments, the second pHis about 4.2.

In some embodiments of the disclosed methods, the benzodiazepine isselected from the group consisting of D1, D1(a), D2, D2(a), DGN462,DGN462(a), D3, D3(a), D4, D4(a), D5, D5(a), D6, and D6(a). In someembodiments, the ADC is selected from the group consisting ofAb-sSPDB-D1, Ab-sSPDB-D1(a), Ab-D2, Ab-D2(a), Ab-sSPDB-DGN462,Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a), Ab-sSPDB-D4, Ab-sSPDB-D4(a),Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1, Ab-Ser-D1(a), Ab-Cys-DGN462,Ab-Cys-DGN462(a), Ab-Ser-DGN462, Ab-Ser-DGN462(a), Ab-Cys-D5,Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a).

In some embodiments, the reversible self association is reduced by about70% to about 80%. In further embodiments, the reversible selfassociation is reduced by about 80% to about 90%. In yet furtherembodiments, the reversible self association is reduced by about 90% to100%.

In some embodiments, the method further comprises lyophilizing theaqueous solution, thereby creating a lyophilized composition. In stillfurther embodiments, the method also comprises reconstituting thelyophilized composition.

In some embodiments, the ADC comprises an antibody selected from thegroup consisting of huMy9-6, huB4, huDS6, huMov19, and huCD37-3. In someembodiments, the ADC comprises a humanized CD123 antibody. In someembodiments, the humanized CD123 antibody is AbX. In some embodiments,the AbX antibody comprises the CDR sequences disclosed herein. In someembodiments, the AbX antibody comprises the heavy chain variable regiondomain sequences and light chain variable region domain sequencesdisclosed herein.

A further aspect of this disclosure is directed to a compositioncomprising (a) an ADC comprising a benzodiazepine and (b) trehalose,wherein the composition has a pH ranges from about 4.0 to about 4.5. Insome embodiments, the composition further comprises sodium succinate. Insome embodiments, the composition further comprises sodium bisulfate. Infurther embodiments, the composition further comprises a surfactant. Insome embodiments, the benzodiazepine is selected from the groupconsisting of D1, D1(a), D2, D2(a), DGN462, DGN462(a), D3, D3(a), D4,D4(a), D5, D5(a), D6, and D6(a). In further embodiments, the ADC isselected from the group consisting of Ab-sSPDB-D1, Ab-sSPDB-D1(a),Ab-D2, Ab-D2(a), Ab-sSPDB-DGN462, Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a),Ab-sSPDB-D4, Ab-sSPDB-D4(a), Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1,Ab-Ser-D1(a), Ab-Cys-DGN462, Ab-Cys-DGN462(a), Ab-Ser-DGN462,Ab-Ser-DGN462(a), Ab-Cys-D5, Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the dynamic light scattering plot of a reversibleself-associating system using huM9-6-sSPDB-DGN462 as an example.

FIGS. 2A and 2B show the SV-AUC distribution of a reversiblyself-associating system using huM9-6-sSPDB-DGN462 as an example.

FIG. 3 shows the changes in hydrodynamic diameter in relation todrug-to-antibody ratio as measured by dynamic light scattering.

FIG. 4 shows the dynamic light scattering plot of different ADCcompositions.

FIG. 5 shows the SV-AUC distribution of different ADC compositions.

FIG. 6 shows the mass spectrometry data for deglycosylated huMOV19-90conjugate.

FIG. 7 shows the mass spectrometry data for deglycosylatedhuMov19-sSPDB-107 conjugate.

FIG. 8 shows the dynamic light scattering plot of different ADCcompositions.

FIG. 9 shows the dynamic light scattering plot of different ADCcompositions.

FIG. 10 shows data obtained from an assessment of RSA for asuccinate-trehalose formulation over a range of pH.

FIG. 11 shows RSA of ADC in 10 mM Sodium succinate, 8% trehalose, 0.01%Polysorbate-20, pH 4.0

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, exemplary suitablemethods and materials are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Definitions

As used herein, the terms “a” or “an” mean one or more unless theseterms are otherwise limited in their use.

As used herein, the term “about” means ±10% of a stated value.

As used herein, the term “subject” means a human or an animal. Exemplaryanimals include, but are not limited to, mammals such as mouse, rat,guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, orrhesus monkey.

As used herein, the term “pharmaceutical formulation” refers to apreparation in a form that may be administered to a subject whileallowing the biological activity of the active ingredient to beeffective.

As used herein, the term “therapeutically effective amount” refers to anamount of a drug that is effective to treat a disease or disorder.

As used herein, “treat”, “treating” or “treatment” refers to thereduction, amelioration, or improvement of a disease or disorder, or thereduction, amelioration, or improvement of at least one symptom of adisease or disorder.

As used herein, the term “irreversible aggregate” refers to non-covalentaggregation typically resulting from a hydrophobic interaction due topartial unfolding.

As used herein, the term “chimeric antibody” refers to an antibodywherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. Typically, the variable region of bothlight and heavy chains corresponds to the variable region of antibodiesderived from one species of mammals (e.g., mouse, rat, rabbit, etc.)with the desired specificity, affinity, and capability while theconstant regions are homologous to the sequences in antibodies derivedfrom another (usually human) to avoid or reduce the chance of elicitingan immune response in that species (e.g., human). In certainembodiments, a chimeric antibody may include an antibody orantigen-binding fragment thereof comprising at least one human heavyand/or light chain polypeptide, such as, for example, an antibodycomprising murine light chain and human heavy chain polypeptides.

As used herein, the term “antigen-binding fragment” of an antibodyrefers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen.

As used herein, “polyclonal” antibodies refer to heterogeneouspopulations of antibody, typically contained in the sera of immunizedanimals.

As used herein, “monoclonal” antibodies refer to homogenous populationsof antibody molecules that are specific to a particular antigen.

As used herein, the terms “linker,” “linking group,” or “linking moiety”refer to a moiety that connects two groups, such as an antibody and acytotoxic compound, together.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Cancer can include ahematological cancer or a solid tumor.

Aggregates and Reversible Self-Association

Development of commercially viable and clinically useful ADCcompositions is complicated by the unpredictable behaviors of differentantibodies and ADCs during formulation. The ability of antibodies andADCs to aggregate and reversibly self-associate can lead to manyundesirable effects commercially and clinically. Aggregation andreversible self-association can lead to reduced potency, decreasedstability, increased toxicity, increased viscosity, discoloration ofsolution, and other undesirable effects. In some circumstances,aggregates can trigger an immune system response, a potential safetyconcern in patients.

Covalent aggregates occur through the formation of a chemical bondbetween at least two monomers. For example, covalent aggregates canresult from disulfide bonds formed between unpaired cysteines on amonomer, or as a result of intermolecular disulfide scrambling, orthrough thioether linking.

In certain instances, aggregation is irreversible. The irreversibleaggregation of immunoglobulins leads to decreased activity orfunctionality of the immunoglobulins in formulations. Irreversibleaggregates can be inhibited by agents such as urea, guanidine, or sodiumdodecyl sulfate (“SDS”), but not through reducing the concentration ofthe antibody.

Reversible self-association (“RSA”) occurs as a result of an ADC'sability to form oligomeric species through weak, non-covalentintermolecular interactions. The amount of these interactions for anygiven ADC in solution depends on a variety of factors, including theantibody itself (e.g., primary and secondary structures) and solutioncharacteristics such as pH, as well as ADC concentration. Furthermore,it has been discovered that the amount and extent of theself-association varies by ADC depending on the characteristics of thecytotoxic compound and the antibody. Cytotoxic compounds that arehydrophobic, insoluble, and/or comprise multiple aromatic rings canincrease RSA. Therefore, more hydrophobic ADCs have an increasedtendency to reversibly self-associate in solution. The ADCsself-associate and attain equilibrium in solution between monomers andin higher ordered oligomeric species.

ADC reversible self-association has numerous detrimental effects informulations. RSA can create problems for manufacturing, stability,delivery, and safety of the ADC in a therapeutic context. From adelivery perspective, RSA can increase the viscosity of a solution,which can impede the plunger of a pre-filled syringe. From a stabilityand safety perspective, RSA can reduce potency (because the oligomericspecies do not function therapeutically) and increase the possibility oftriggering an immune response.

Researchers have several methods at their disposal to assess the amountof ADC monomer in solution. For example, the amount of ADC monomer insolution can be measured by size exclusion chromatography (both SEC andSEC-MS) and sedimentation velocity (SV). Under SEC analysis, ADCaggregates elute more quickly than ADC monomers that are smaller andable to travel deeper into the pores of the SEC packing material. SECcan provide good separation between aggregates and monomers, therebyproviding a good estimation of the amount of monomer in the solution.

As explained in greater detail in the Examples below, SV analyzes thebehavior of the ADC in solution by applying angular acceleration to thesolution (generally through centrifugation) to cause the ADCs tosediment. Generally, larger particles, e.g., ADC aggregates orreversibly self-associated oligomers, sediment more quickly. Therefore,SV can be used to assess the amount of ADC monomer in solution becausethe aggregates and oligomers sediment more quickly than the monomers.

In some instances, SEC or SEC-MS and SV may give different monomerpercentages for the same antibody. Such discrepancies can indicate thepresence of reversibly self-associating monomers. Moreover, changes inconcentration and solution characteristics typically reduce RSA, but maynot affect the amount of either covalent or irreversible aggregatespresent.

Multiangle Light Scattering (“MALS”) can be used to determine the amountof reversibly associated oligomers in a given solution based on how themonomers and the oligomers of different order scatter light. As theconcentration of an antibody in solution increases, MALS typicallydetects the increase of a species of higher molecular weight, e.g., anoligomer, than the monomer. This increase indicates an increase in RSA.

Dynamic Light Scattering (“DLS”) can also be used to determine theexistence and extent of RSA in a solution. DLS involves measuring thetime-dependent change in intensity of light scattered by a species insolution. Typically, DLS-measuring instruments yield the hydrodynamicdiameter of a particle. As the concentration of an antibody in solutionincreases, a DLS-measuring instrument will detect an increased presenceof species having greater hydrodynamic diameters, e.g., oligomers. Thisincrease indicates an increase in RSA. DLS-measuring instruments canalso be used to determine the diffusion coefficients of the species insolution. Diffusion coefficients decrease with increased antibodyconcentration, indicating the existence of RSA.

ADCs

This disclosure is directed to ADCs, compositions comprising ADCs,methods of treating, methods of formulating ADC compositions, andmethods of reducing RSA in ADCs. ADCs comprise an antibody, or anantibody fragment, conjugated to a cytotoxic compound. In someembodiments, the cytotoxic compound is conjugated to an antibody via alinker. In other embodiments, the cytotoxic compound is linked directlyto the antibody. The types of antibodies, linkers, and cytotoxiccompounds encompassed by this disclosure are described below.

Antibodies

Disclosed herein are compositions that comprise antibodies andantigen-binding fragments thereof. Antibodies are large glycoproteinsthat can exist as soluble and membrane-bound forms and comprise fivenatural isotypes—IgA, IgD, IgE, IgG, and IgM, based on the identity oftheir heavy-chain constant domains referred to as alpha, delta, epsilon,gamma, and mu, respectively. The different classes of immunoglobulinshave different and well-known subunit structures and three-dimensionalconfigurations. As one of ordinary skill in the art will recognize, thedisclosed compositions can comprise polyclonal antibodies and monoclonalantibodies. In particular embodiments, the antibodies compriseantibodies such as multispecific antibodies such as bispecificantibodies, chimeric antibodies, humanized antibodies, and humanantibodies. The compositions can also comprise antibodies that have oneor more conservative or non-conservative amino acid substitutions.Furthermore, the compositions can comprise modified glycosylation at oneor more amino acid residues. Such modified antibodies or bindingfragments fall within the scope of the compositions disclosed herein solong as the modified antibodies exhibit the desired biological activity.

As used herein, a “humanized” antibody is one in which thecomplementarity-determining regions (CDRs) of a mouse monoclonalantibody, which form the antigen binding loops of the antibody, aregrafted onto the framework of a human antibody molecule or where thevariable domains of the framework of a murine antibody have beenresurfaced (i.e., the exposed residues are replaced with the residuesthat are present in the corresponding positions of human antibodies).See, e.g., Roguska et. al, Protein Engineering, Vol 9. No. 10, pp.895-904, 1996.

Exemplary antibodies include humanized monoclonal antibodies, examplesof which include, huMy9-6, huB4, huDS6, huMov19, and huCD37-3. Exemplaryantibodies also include humanized CD123 antibodies, exemplary sequencesof which are described in U.S. application publication no.US20170029514A1 and PCT application publication no. WO2017004026 and arereferred to herein as “AbX”.

Specific examples of humanized CD123 antibodies (AbX) described in U.S.application publication no. US20170029514A1 and PCT applicationpublication no. WO2017004026 and included in the AbX embodimentsdescribed herein are:

Humanized CD123 antibodies that include the following heavy chainvariable region CDR amino acid sequences:

V_(H) CDR1: SSIMH (SEQ ID NO: 1) V_(H) CDR2: YIKPYNDGTKYNEKFKG(SEQ ID NO: 2) V_(H) CDR3: EGGNDYYDTMDY (SEQ ID NO: 3)

Humanized CD123 antibodies that include the following light chainvariable region CDR amino acid sequences:

V_(L) CDR1: RASQDINSYLS (SEQ ID NO: 4) V_(L) CDR2: RVNRLVD(SEQ ID NO: 5) V_(L) CDR3: LQYDAFPYT (SEQ ID NO: 6)

Humanized anti-CD123 antibodies that include the following heavy chainvariable region amino acid sequences:

AbX₁: (SEQ ID NO: 7) Q(F/V)QLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSS AbX₂: (SEQ ID NO: 8)QVQLVQSGAEVKKPGASVKVSCKASGYGFTSSIMHWVRQAPGQGLEWMGYIKPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREG GNDYYDTMDYWGQGTLVTVSS

Humanized anti-CD123 antibodies that include the following light chainvariable region amino acid sequences:

AbX₁: (SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQ GTKVEIKR AbX₂:(SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQDINSYLAWFQQKPGKAPKSLIYRVNRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDAFPYTFGQ GTKVEIKR

In other specific embodiments, the anti-CD123 antibody orantigen-binding fragment thereof comprises an engineered Cys residue(e.g., C442); an immunoglobulin heavy chain variable domain at leastabout 90%, 95%, 99% or 100% identical toQXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGT LVTVSS (SEQ IDNO: 7); and an immunoglobulin light chain variable region having theamino acid sequence at least about 90%, 95%, 99% or 100% identical toDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKR (SEQ ID NO: 9). Incertain embodiments, Xaa, the second residue from the N-terminus of SEQID NO: 7, is Phe. In other embodiments, Xaa is Val.

In other specific embodiments, the anti-CD123 antibody orantigen-binding fragment thereof comprises an engineered Cys residue(e.g., C442); an immunoglobulin heavy chain variable domain at leastabout 90%, 95%, 99% or 100% identical toQVQLVQSGAEVKKPGASVKVSCKASGYGFTSSIMHWVRQAPGQGLEWMGYIKPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQ GTLVTVSS (SEQID NO: 8); and an immunoglobulin light chain variable region having theamino acid sequence at least about 90%, 95%, 99% or 100% identical toDIQMTQSPSSLSASVGDRVTITCRASQDINSYLAWFQQKPGKAPKSLIYRVNRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKR (SEQ ID NO: 10).

Humanized anti-CD123 antibodies that include the following heavy chainamino acid sequences:

AbX₁: (SEQ ID NO: 11) Q(F/V)QLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG AbX_(1C):(SEQ ID NO: 12) Q(F/V)QLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLC LSPG AbX₂:(SEQ ID NO: 13) QVQLVQSGAEVKKPGASVKVSCKASGYGFTSSIMHWVRQAPGQGLEWMGYIKPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Humanized anti-CD123 antibodies that include the following light chainamino acid sequences:

AbX₁: (SEQ ID NO: 14) DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC AbX₂:(SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITCRASQDINSYLAWFQQKPGKAPKSLIYRVNRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

In other specific embodiments, the anti-CD123 antibody orantigen-binding fragment thereof comprises an engineered Lys residue; animmunoglobulin heavy chain variable domain at least about 90%, 95%, 99%or 100% identical toQ(F/V)QLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11); and animmunoglobulin light chain variable region having the amino acidsequence at least about 90%, 95%, 99% or 100% identical toDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14). In certainembodiments, Xaa, the second residue from the N-terminus of SEQ ID NO:11, is Phe. In other embodiments, Xaa is Val.

In other specific embodiments, the anti-CD123 antibody orantigen-binding fragment thereof comprises an immunoglobulin heavy chainvariable domain at least about 90%, 95%, 99% or 100% identical toQ(F/V)QLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG (SEQ ID NO: 12); and animmunoglobulin light chain variable region having the amino acidsequence at least about 90%, 95%, 99% or 100% identical toDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14). In certainembodiments, Xaa, the second residue from the N-terminus of SEQ ID NO:12, is Phe. In other embodiments, Xaa is Val.

In other specific embodiments, the anti-CD123 antibody orantigen-binding fragment thereof comprises an immunoglobulin heavy chainvariable domain at least about 90%, 95%, 99% or 100% identical toQVQLVQSGAEVKKPGASVKVSCKASGYGFTSSIIVIHWVRQAPGQGLEWMGYIKPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13); and animmunoglobulin light chain variable region having the amino acidsequence at least about 90%, 95%, 99% or 100% identical toDIQMTQSPSSLSASVGDRVTITCRASQDINSYLAWFQQKPGKAPKSLIYRVNRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15).

Exemplary sequences for huDS6 are described in U.S. Pat. No. 7,834,155and International Pat. Appl. Publication Nos.: WO2005/009369 andWO2007/024222, which are incorporated herein by reference in theirentireties. Detailed sequences for huMov19 are described in U.S. Pat.Nos. 8,557,966 and 8,709,432 and International Pat. Appl. PublicationNos.: WO2011/106528, which are incorporated herein by reference in theirentireties. Exemplary sequences for the huMy9-6 heavy chain variableregion portion are described in U.S. Patent Publication No. 20060177455,which is incorporated herein by reference in its entirety. Exemplarysequences for the huMy9-6 light chain variable region portion are knownin the art and described in U.S. Pat. Nos. 7,557,189, 7,342,110,8,119,787 and 8,337,855, which are incorporated herein by reference intheir entireties. Exemplary sequences for huCD37-3 are described in U.S.Pat. No. 8,765,917 and International Pat. Appl. Publication No.WO2011/112978, which are incorporated herein by reference in theirentireties. Exemplary sequences for huB4 is described in InternationalPat. Appl. Publication No. WO2012/156455, which is incorporated hereinby reference in its entirety.

Additional exemplary antibodies include antibodies that target specificantigens. Examples include antibodies that target CD33, CD19, CD37, CA6,or FOLR1. Further, antibodies that target CD123 are also includedherein.

Generally, the term “humanized antibody” refers to forms of non-human(e.g., murine) antibodies that are specific immunoglobulin chains,chimeric immunoglobulins, or fragments thereof that contain minimalnon-human (e.g., murine) sequences. Typically, humanized antibodies arehuman immunoglobulins in which residues from the complementarydetermining region (CDR) are replaced by residues from the CDR of anon-human species (e.g., mouse, rat, rabbit, hamster) that have thedesired specificity, affinity, and capability (Jones et al, Nature321:522-525, 1986; Riechmann et al, Nature 332:323-327, 1988; Verhoeyenet al, Science 239:1534-1536, 1988).

Antibodies can be humanized using a variety of other techniquesincluding CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos.5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0519 596; Padlan E. A., 1991, Molecular Immunology 28(4/5):489-498;Studnicka G. M. et al., 1994, Protein Engineering 7(6):805-814; RoguskaM. A. et al., 1994, PNAS 91:969-973), and chain shuffling (U.S. Pat. No.5,565,332). Human antibodies can be made by a variety of methods knownin the art including phage display methods. See also U.S. Pat. Nos.4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International Pat.Appl. Publication Nos.: WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said referencesincorporated by reference in their entireties).

In some instances, the F_(v) framework region (FR) residues of a humanimmunoglobulin are replaced with the corresponding residues in anantibody from a non-human species that has the desired specificity,affinity, and capability. The humanized antibody can be further modifiedby the substitution of additional residues either in the F_(v) frameworkregion and/or within the replaced non-human residues to refine andoptimize antibody specificity, affinity, and/or capability. In general,the humanized antibody will comprise substantially all of at least one,and typically two or three, variable domains containing all orsubstantially all of the CDR regions that correspond to the non-humanimmunoglobulin whereas all or substantially all of the FR regions arethose of a human immunoglobulin consensus sequence. The humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region or domain (F_(c)), typically that of a humanimmunoglobulin. Examples of methods used to generate humanizedantibodies are described in U.S. Pat. Nos. 5,225,539 and 5,639,641,Roguska et al, Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994; andRoguska et al, Protein Eng. 9(10):895-904, 1996 (all incorporated hereinby reference). In some embodiments, a “humanized antibody” is aresurfaced antibody. In some embodiments, a “humanized antibody” is aCDR-grafted antibody.

In addition, the antibody can be a chimeric antibody.

The disclosed compositions can comprise antigen-binding fragments suchas antibody fragments (such as Fab, Fab′, F(ab)2, and Fv fragments) orsingle chain Fv (scFv) mutants. In other embodiments, the bindingfragments are attached to a separate protein, peptide, or oligopeptideto form a fusion protein. In certain embodiments, the fusion proteincomprises an antigen-determination portion of an antibody fused to a oneor more peptides, oligopeptides, or polypeptides.

One of ordinary skill in the art will appreciate that the selection ofan appropriate antibody will depend upon the cell population to betargeted. In this regard, the type and number of cell surface molecules(i.e., antigens) that are selectively expressed in a particular cellpopulation (typically a diseased cell population) will inform theselection of an appropriate antibody for use in the disclosedcompositions. Cell surface expression profiles are known for a widevariety of cell types, including tumor cell types, or, if unknown, canbe determined using routine molecular biology and histochemistrytechniques.

As noted herein, the antibodies can be polyclonal or monoclonal.Monoclonal antibodies are typically produced by a single clone of Blymphocytes (“B cells”). Monoclonal antibodies may be obtained using avariety of techniques known to those skilled in the art, includingstandard hybridoma technology (see, e.g., Köhler and Milstein, Eur. J.Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: ALaboratory Manual, CSH Press (1988), and C. A. Janeway et al. (eds.),Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). Inbrief, the hybridoma method of producing monoclonal antibodies typicallyinvolves injecting any suitable animal, typically a mouse, with anantigen (i.e., an “immunogen”). The animal is subsequently sacrificed,and B cells isolated from its spleen are fused with human myeloma cells.A hybrid cell is produced (i.e., a “hybridoma”), which proliferatesindefinitely and continuously secretes high titers of an antibody withthe desired specificity in vitro. Any appropriate method known in theart can be used to identify hybridoma cells that produce an antibodywith the desired specificity. Such methods include, for example,enzyme-linked immunosorbent assay (ELISA), Western blot analysis, andradioimmunoassay. The population of hybridomas is screened to isolateindividual clones, each of which secretes a single antibody species tothe antigen. Because each hybridoma is a clone derived from fusion witha single B cell, all the antibody molecules it produces are identical instructure, including their antigen binding site and isotype. Monoclonalantibodies also may be generated using other suitable techniquesincluding EBV-hybridoma technology (see, e.g., Haskard and Archer, J.Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., MethodsEnzymol., 121, 140-67 (1986)), or bacteriophage vector expressionsystems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)). Toprepare monoclonal antibody fragments, recombinant methods typically areemployed.

The monoclonal antibody can be isolated from or produced in any suitableanimal. In some embodiments, the antibody is produced in a mammal. Insome embodiments, the mammal is a mouse. In some embodiments, the mammalis a human. Methods for producing an antibody in mice are well known tothose skilled in the art and are described herein. With respect to humanantibodies, one of ordinary skill in the art will appreciate thatpolyclonal antibodies can be isolated from the sera of human subjectsvaccinated or immunized with an appropriate antigen. Alternatively,human antibodies can be generated by adapting known techniques forproducing human antibodies in non-human animals such as mice (see, e.g.,U.S. Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Although effective for human therapeutic use, human antibodies,particularly human monoclonal antibodies, typically are more difficultto generate than mouse monoclonal antibodies. Mouse monoclonalantibodies, however, induce a rapid host antibody response whenadministered to humans, which can reduce the therapeutic or diagnosticpotential of an ADC. To circumvent these complications, a monoclonalantibody preferably is not recognized as “foreign” by the human immunesystem. To this end, phage display can be used to generate the antibody.In this regard, phage libraries encoding antigen-binding variable (V)domains of antibodies can be generated using standard molecular biologyand recombinant DNA techniques (see, e.g., Sambrook et al. (eds.),Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring HarborLaboratory Press, New York (2001)). Phage encoding a variable regionwith the desired specificity are selected for specific binding to thedesired antigen, and a complete human antibody is reconstitutedcomprising the selected variable domain. Nucleic acid sequences encodingthe reconstituted antibody are introduced into a suitable cell line,such as a myeloma cell used for hybridoma production, such that humanantibodies having the characteristics of monoclonal antibodies aresecreted by the cell (see, e.g., Janeway et al., supra, Huse et al.,supra, and U.S. Pat. No. 6,265,150). Alternatively, monoclonalantibodies can be generated from mice that are transgenic for specifichuman heavy and light chain immunoglobulin genes. Such methods are knownin the art and described in, for example U.S. Pat. Nos. 5,545,806 and5,569,825, and Janeway et al., supra. In some embodiments, the antibodyis a humanized antibody. Owing to the similarity of the frameworks ofmouse and human antibodies, it is generally accepted in the art thatthis approach produces a monoclonal antibody that is antigenicallyidentical to a human antibody but binds the same antigen as the mousemonoclonal antibody from which the CDR sequences were derived. Methodsfor generating humanized antibodies are known in the art and aredescribed in detail in, for example, Janeway et al., supra, U.S. Pat.Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No. 0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodies can alsobe generated using the antibody resurfacing technology described in U.S.Pat. No. 5,639,641, Pedersen et al., J. Mol. Biol., 235, 959-973 (1994),Roguska et al., Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994; andRoguska et al, Protein Eng. 9(10):895-904, 1996.

Antibody fragments that have at least one antigen-binding site, and thusrecognize and bind to at least one antigen or receptor present on thesurface of a target cell, also are within the scope of this disclosure.In this respect, proteolytic cleavage of an intact antibody molecule canproduce a variety of antibody fragments that retain the ability torecognize and bind antigens. For example, limited digestion of anantibody molecule with the protease papain typically produces threefragments, two of which are identical and are referred to as the Fabfragments, as they retain the antigen binding activity of the parentantibody molecule. Cleavage of an antibody molecule with the enzymepepsin normally produces two antibody fragments, one of which retainsboth antigen-binding arms of the antibody molecule, and is thus referredto as the F(ab′)2 fragment. A single-chain variable region fragment(sFv) antibody fragment, which consists of a truncated Fab fragmentcomprising the variable (V) domain of an antibody heavy chain linked toa V domain of a light antibody chain via a synthetic peptide, can begenerated using routine recombinant DNA technology techniques (see,e.g., Janeway et al., supra). Similarly, disulfide-stabilized variableregion fragments (dsFv) can be prepared by recombinant DNA technology(see, e.g., Reiter et al., Protein Engineering, 7, 697-704 (1994)).Antibody fragments of the present disclosure, however, are not limitedto these exemplary types of antibody fragments. Any suitable antibodyfragment that recognizes and binds to a desired cell surface receptor orantigen can be employed. Antibody-antigen binding can be assayed usingany suitable method known in the art, such as, for example,radioimmunoassay (MA), ELISA, Western blot, immunoprecipitation, andcompetitive inhibition assays (see, e.g., Janeway et al., supra, andU.S. Patent Application Publication No. 2002/0197266 A1).

Linkers

Aspects of the compositions disclosed herein comprise antibodies andantigen-binding fragments thereof attached to a linker. Any suitablelinker can be used with the ADCs of the present disclosure as long asthe linker does not prevent the antibody from binding to its target oreliminate a cytotoxic compound's cytotoxicity. Typically, a linker issubstantially inert under conditions for linking two groups. In someembodiments, a linker moiety comprises two reactive groups, such thatone reactive group can be first reacted with the cytotoxic compound toprovide a compound bearing the linker moiety and a second reactivegroup, which can then react with an antibody. Alternatively, onereactive group of the linker moiety can be first reacted with anantibody to provide an antibody and a linker moiety and a secondreactive group, which can then react with a cytotoxic compound.

Exemplary linkers include, but are not limited to, disulfide linkers,thioether linkers, amide bonded linkers, peptidase-labile linkers,acid-labile linkers, and esterase-labile linkers. In some embodiments,the linker is cleavable. Exemplary cleavable linkers include, but arenot limited to, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), or N-succinimidyl4-methyl-4-[2-(5-nitro-pyridyl)-dithio]pentanoate (SMNP). In someembodiments, the linker is non-cleavable. Exemplary non-cleavablelinkers include, but are not limited to, 2-iminothiolane, acetylsuccinicanhydride, and succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(SMCC). Other exemplary linkers, such as CX1-1 (as described in U.S.Pat. Publication No. US20120253021, which is incorporated herein byreference) and acetylsuccinic anhydride, can be used as cleavable ornon-cleavable linkers.

In some embodiments, the linking moiety contains a chemical bond thatallows for the release of the cytotoxic compound at a particular site.Suitable chemical bonds are well known in the art and include disulfidebonds, thioether bonds, acid labile bonds, photolabile bonds, peptidaselabile bonds and esterase labile bonds (see for example U.S. Pat. Nos.5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497;7,276,499; 7,368,565; 7,388,026 and 7,414,073). Other suitable linkersinclude non-cleavable linkers, such as those described in are describedin detail in U.S. publication number 20050169933, or charged linkers orhydrophilic linkers and are described in US 2009/0274713, US2010/01293140 and WO 2009/134976, each of which is expresslyincorporated herein by reference.

Cytotoxic Compounds

Aspects of this disclosure are directed to several cytotoxic compounds.These cytotoxic compounds can induce cytotoxicity in cells. When coupledto any of the above-described antibodies to form the ADCs of thisdisclosure, these cytotoxic compounds can be delivered directly totargeted cells. As a result of normal pharmacologic clearancemechanisms, an antibody employed in an ADC contacts and binds to targetcells only in limited amounts. Therefore, the cytotoxic agent employedin the conjugate must be highly cytotoxic such that cell killingsufficient to elicit a therapeutic effect occurs.

The cytotoxic compounds of this disclosure comprise benzodiazepines. Insome embodiments, the cytotoxic compound is a pyrrolobenzodiazepine. Insome embodiments, the cytotoxic compound is a indolinobenzodiazepine. Insome embodiments, the cytotoxic compound is a compound in Table 1.DGN462 is described, for example, in U.S. Pat. No. 8,765,740, which isincorporated herein by reference in its entirety. Compound D3 isdescribed, for example, in U.S. Pat. Nos. 8,426,402, 8,809,320 and8,802,667, which are incorporated herein by reference in their entirety.Compounds D1, D2, and D4 are described, for example, in U.S. ApplicationPublication Numbers US20160106863A1 and US20160082114A1, which are bothincorporated herein by reference, and “Antibody-Drug Conjugates (ADCs)of Indolino-Benzodiazepine DNA-Alkylating Agents”, 2015 AACR, Abstractnumber 652, which is incorporated herein by reference.

TABLE 1 Compound Name Compound D1

D1(a)

D2

D2(a)

DGN462

DGN462(a)

D3

D3(a)

D4

D4(a)

D5

D5(a)

D6

D6(a)

The benzodiazepines, including the compounds in Table 1, are linked toantibodies and antigen-binding fragments thereof with the linkersdescribed herein. In some embodiments of the ADCs, the benzodiazepinesmay be Cys-linked. In other embodiments, the benzodiazepines may beSer-linked.

This disclosure is also directed to variations of the compounds in Table1, such as modification of a compound in Table 1 by sulfonation. Othervariations of the compounds in Table 1 are readily apparent to those ofordinary skill in the art. Such variations are encompassed by thisdisclosure.

Increased RSA in ADCs

ADCs comprising benzodiazepines are shown herein to exhibit increasedRSA. It is believed that the increased RSA results from the increasedhydrophobic interactions from the benzodiazepines resulting inadditional reversible intermolecular interactions. As demonstrated belowin the Examples, an increase in the drug load (the “DAR” ordrug-to-antibody ratio) results in increased RSA. Compositions withhigher DARs have higher amounts of benzodiazepines per antibody. Thebenzodiazepines are hydrophobic and insoluble and comprise multiplearomatic rings. Thus, it is believed, the benzodiazepines interact withother components in an ADC. These additional reversible intermolecularinteractions result in increased RSA for the ADCs disclosed herein.Therefore, the amount of RSA increases as the drug load increases. As aresult, it is even more difficult to develop pharmaceutical formulationsfor ADCs comprising hydrophobic molecules (e.g., benzodiazepines orindolinobenzodiazepines) of this disclosure.

Reduced RSA Compositions

It has been surprisingly discovered that certain small hydrophobicmolecules inhibit or reduce RSA in compositions comprising the ADCs ofthis disclosure. These small molecules fall into two classes: (1) aminoacids with hydrophobic side chains, including proline, alanine, leucine,isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine;and (2) betaines, small neutral molecules with a positively chargedcationic functional group and a negatively charged functional group. Thecationic functional group and negatively charged functional group neednot be adjacent. The cationic functional groups include onium ions suchas quaternary ammonium and quaternary phosphonium. The negativelycharged functional groups include carboxylate, sulfite, and phosphite.An exemplary betaine is trimethylglycine. Historically, the term betainereferred to trimethlyglycine. Therefore, depending on the context asused herein, the term “betaine” can refer to betaines generally or totrimethlyglycine specifically.

One aspect of this disclosure is directed to a composition comprising:(a) an ADC comprising a benzodiazepine; and (b) a small hydrophobicmolecule selected from the group consisting of betaines and or aminoacids with hydrophobic side chains. In some embodiments, the smallhydrophobic molecule is an amino acid with a hydrophobic side chain. Insome embodiments, the small hydrophobic molecule is a betaine. In someembodiments, the small hydrophobic molecule is trimethlyglycine. In someembodiments, the antibody is selected from the group consisting ofhuMy9-6, huB4, huDS6, huMov19, and huCD37-3. In other embodiments, theantibody is a humanized CD123 antibody. In certain embodiments, theantibody is AbX. In some embodiments, the benzodiazepine is anindolinobenzodiazepine. In some embodiments, the benzodiazepine is acompound in Table 1. In some embodiments, the ADC is an ADC in Table 2.

TABLE 2 Conjugate Name Conjugate Structure Ab-sSPDB- D1

Ab-sSPDB- D1(a)

Ab-D2

Ab-D2(a)

Ab-sSPDB- DGN462

Ab-sSPDB- DGN462(a)

Ab-D3

Ab-D3(a)

Ab-sSPDB- D4

Ab-sSPDB- D4(a)

Ab-Cys-D1

Ab-Cys- D1(a)

Ab-Ser-D1

Ab-Ser- D1(a)

Ab-Cys- DGN462

Ab-Cys- DGN462(a)

Ab-Ser- DGN462

Ab-Ser- DGN462(a)

Ab-Cys-D5

Ab-Cys- D5(a)

Ab-Ser-D6

Ab-Ser- D6(a)

Wherein, in Table 2, r is an integer from 1 to 10; Ab-NH is an antibodycovalently linked to the compound through a lysine; Ser indicates anantibody linked to the compound through an N-terminal serine; Cysindicates an antibody linked to the compound through a cysteine; and Mis H⁺, Na⁺, K⁺, or any pharmaceutically acceptable cation.

This disclosure is also directed to other variations in the linker ofthe ADCs in Table 2, that are readily apparent to those of ordinaryskill in the art. For example, the SO₃ M group shown on the linker canbe substituted with ‘H’ to obtain an ADC wherein the antibody Ab islinked via SPDB linker to the cytotoxic compounds D1, D1(a), D2, D2(a),DGN462, DGN462(a), D3, D3(a), D4, D4(a), D5, D5(a), D6, and D6(a),respectively. Such variations and similar variations are encompassed bythis disclosure.

In some embodiments, RSA is eliminated in the disclosed compositions. Insome embodiments, RSA is decreased by about 90% to 100% in the disclosedcompositions. In certain embodiments, RSA is decreased by about 80% toabout 90% in the disclosed compositions. In some embodiments, RSA isdecreased by about 70% to about 80% in the disclosed compositions. Infurther embodiments, RSA is decreased by about 60% to about 70% in thedisclosed compositions. In still further embodiments, the RSA isdecreased by about 50% to about 60% in the disclosed compositions. Inyet further embodiments, RSA is decreased by about 40% to about 50% inthe disclosed compositions. In some embodiments, RSA is decreased byabout 30% to about 40% in the disclosed compositions.

Previous formulations for antibodies or ADCs have been buffered to a pHof approximately 5-6.5 in order to maintain the structure and stabilityof the antibody or the ADC. Surprisingly, we have discovered that pH canaffect the amount and extent of RSA in a solution and, specifically,that a lower than expected pH of approximately 4-4.5, can reduce RSA foran ADC. Another aspect of this disclosure is directed to compositionscomprising ADCs comprising a benzodiazepine, wherein the composition hasa low pH. It is believed that a lower pH allows for a higher amount ofH⁺ ions that can interact non-covalently with the antibodies in solutionand inhibit the antibodies' ability to form oligomeric species throughintermolecular interactions, thereby reducing RSA. In some embodiments,the composition has a pH between about 4.0 to about 4.5. One aspect ofthis disclosure is directed to a composition having a pH between about4.0 and about 4.5 and comprising a betaine and an ADC comprising abenzodiazepine. In some embodiments, the betaine is trimethlyglycine.Another aspect of this disclosure is directed to a composition having apH between 4.0 and 4.5 and comprising an amino acid with a hydrophobicside chain and an ADC comprising a benzodiazepine. In some embodiments,the benzodiazepine is a compound in Table 1. In some embodiments, theADC is an ADC in Table 2.

The compositions of this disclosure are formulated to be acceptable forpharmaceutical use, such as, for example, administration to a human inneed thereof. In some embodiments, the ADC is formulated into acomposition comprising a physiologically acceptable carrier (e.g.,excipient or diluent). Physiologically acceptable carriers are wellknown and are readily available, and include buffering agents,anti-oxidants, bacteriostats, salts, and solutes that render thecomposition isotonic with the blood or other bodily fluid of the humanpatient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers(e.g., surfactants), and preservatives. The choice of carrier will bedetermined, at least in part, by the location of the target tissueand/or cells, and the particular method used to administer thecomposition. Examples of suitable carriers and excipients for use in ADCpharmaceutical formulations are disclosed in, for example, International(PCT) Patent Application Nos. WO 00/02587, WO 02/060955, and WO02/092127, and Ghetie et al., J. Immunol. Methods, 112, 267-277 (1988).

Pharmaceutically acceptable buffering agents may be used in connectionwith the disclosed compositions. In some embodiments, succinate may beused as a buffering agent in connection with the disclosed compositions.In other embodiments, citrate may be used as a buffering agent inconnection with the disclosed compositions. In some embodiments, sodiumbisulfate may be used in addition to succinate or citrate. Otherexemplary buffering agents that may be used with the disclosedcompositions include acetate and phosphate. The buffering agent may bepresent in the compositions of this disclosure in any suitableconcentration, so long as sufficient stability of the composition isachieved under the desired conditions. In this regard, the concentrationof the buffering agent in the composition is about 2 mM to about 50 mM(e.g., about 2-10 mM, about 10-20 mM, about 20-30 mM, about 30-40 mM, orabout 40-50 mM). In some embodiments, the concentration of the bufferingagent in the composition is about 5-15 mM (e.g., about 10 mM). In someembodiments, the buffering agent is sodium succinate or sodium acetate.In some embodiments, the buffering agent is sodium citrate. Thebuffering agent typically is present in the disclosed compositions suchthat the pH is maintained within a desired range.

The compositions of this disclosure also optionally contain asurfactant. Any suitable surfactant can be used. Suitable surfactantsare well known to those skilled in the art. In some embodiments, thesurfactant is a polysorbate. In some embodiments, the surfactant ispolysorbate 20 or polysorbate 80. The surfactant may be present in thecompositions of this disclosure in any suitable concentration, so longas sufficient stability of the composition is achieved under the desiredconditions. In this regard, the concentration of the surfactant in thecomposition is about 0.002% to about 0.1% wt./vol. (e.g., about0.002-0.01%, about 0.005-0.02%, or about 0.01-0.1% wt./vol.) of thetotal volume of the composition. In some embodiments, the concentrationof the surfactant in the composition is about 0.005-0.02% wt./vol.(e.g., about 0.01% wt./vol.) of the total volume of the composition.

The composition of this disclosure can further be stabilized by theaddition of sugar. In some embodiments, the sugar is sucrose ortrehalose. In some embodiments, the concentration of sucrose ortrehalose in the composition is about 0.1% to about 10% wt./vol. (e.g.,about 0.1-1%, about 2-5%, or about 7-10% wt./vol.) of the total volumeof the composition. The composition can also further comprise bulkingagents. In some embodiments, the bulking agent is mannitol. In otherembodiments, the bulking agent is glycine.

The compositions of this disclosure can be lyophilized. Lyophilizationrefers to freeze drying under a vacuum. Lyophilization typically isaccomplished by freezing a particular formulation such that the solutesare separated from the solvent(s). The solvent is then removed bysublimation (i.e., primary drying) and next by desorption (i.e.,secondary drying). When the compositions of this disclosure arelyophilized and then reconstituted, RSA is still reduced or inhibited.Thus, although the small hydrophobic molecule can be added tocompositions prior to lyophilization, the benefits of reduced orinhibited RSA are still realized in the compositions that arereconstituted after lyophilization.

In order to prevent degradation of the composition during freezing anddrying, the lyophilized composition optionally further comprises acryoprotectant. In some embodiments, the cryoprotectant is an amorphouscryoprotectant. The term “cryoprotectant,” as used herein, refers to anexcipient that protects unstable molecules during freezing. Suitablecryoprotectants for use in the compositions of this disclosure are knownto those skilled in the art, and include, for example, glycerol,dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), dextran, glucose,trehalose, and sucrose. In some embodiments, the cryoprotectant issucrose. The cryoprotectant may be present in the lyophilizedcomposition in any suitable amount. In some embodiments, the lyophilizedcomposition comprises about 0.5 mg to about 5 mg (e.g., about 0.5 mg toabout 2 mg) of the cryoprotectant per mg of the conjugate (e.g., about0.8 mg cryoprotectant per mg of the conjugate. In some embodiments, thelyophilized composition comprises about 2 mg cryoprotectant per mg ofthe conjugate. In some embodiments, the lyophilized compositioncomprises about 4 mg cryoprotectant per mg of the conjugate. In someembodiments, the cryoprotectant is sucrose and the lyophilizedcomposition comprises about 0.5 mg to about 2 mg (e.g., about 1 mg)sucrose per mg of the conjugate

Lyophilization methods are well known in the art and are described in,for example, Wang, W., Int. J. Pharm., 203, 1-60 (2000). For example,the lyophilized compositions of this disclosure can be produced using alyophilization cycle comprising the following steps: (1) pre-cooling ata shelf temperature of 4° C. and ambient chamber pressure for 2.5 hours,(2) freezing at a shelf temperature of −50° C. and ambient chamberpressure for 14 hours, (3) glycine recrystallization at a shelftemperature of −20° C. and ambient chamber pressure for 6 hours, (4)re-freezing at a shelf temperature of −50° C. and ambient chamberpressure for 16 hours, (5) primary drying at a shelf temperature of −13°C. and 100 mTorr of pressure for 24 hours, (6) secondary drying at ashelf temperature of 24° C. and 100 mTorr of pressure for 10 hours, and(7) stopper phase at a shelf temperature of 24° C. and ambient chamberpressure. However, lyophilized compositions of this disclosure are notlimited to compositions produced by the above-described method. Indeed,any suitable lyophilization method can be used to produce thelyophilized compositions of this disclosure, and it will be apparent tothose skilled in the art that the chosen lyophilization parameters(e.g., drying times) will vary depending on a variety of factors,including the volume of the solution to be lyophilized.

The compositions of this disclosure are advantageous over the prior artformulations for many reasons. The increase in the amount of monomerresults in compositions of increased potency. Efficacy is increasedbecause each composition delivers greater therapeutic effect per dose.This is advantageous because it reduces the number of doses thatsubjects need.

In addition to the increased potency, the compositions of thisdisclosure also decrease toxicity, hence improving patient safety. Thecompositions of this disclosure deliver more of the cytotoxic compoundsto the targeted sites by virtue of the reduced RSA, thereby reducing theamount of cytotoxic compounds that can interact with non-targeted sites.Furthermore, the reduced RSA decreases the viscosity of a solution,thereby improving the efficacy of some modes of administration becausethe disclosed compositions are less likely to clog or impede the plungerof a syringe.

Methods of Treating

This disclosure is also directed to methods of treating cancer in asubject comprising administering to the subject a composition comprising(a) an effective amount of an ADC comprising a benzodiazepine and (b) asmall hydrophobic molecule selected from the group consisting ofbetaines and amino acids with hydrophobic side chains, wherein the ADCis cytotoxic in one or more cells, thereby treating the cancer. In someembodiments, the composition has a pH of about 4.0 to about 4.5. Thedescriptions of the ADCs comprising a benzodiazepine, the smallhydrophobic molecules, excipients (e.g., buffering agents, surfactants,sugars, etc.), and other components described herein are also applicableto the compositions that are used in the methods of treating.

While any suitable means of administering the composition to a subjectcan be used, in some embodiments, the disclosed compositions areadministered to a human via injection. In some embodiments, thedisclosed compositions are administered to a human via infusion. As usedherein, the term “injection” refers to the forceful introduction of thedisclosed compositions into a target tissue of the human. As usedherein, the term “infusion” refers to the introduction of the disclosedcompositions into a tissue, e.g., a vein, of the human. The compositioncan be administered to the human by any suitable route. In someembodiments, the compositions are administered to the humanintravenously or intraperitoneally. In some embodiments, administrationis intratumoral. When the composition is administered by injecting, anysuitable injection device can be used to administer the composition. Forexample, the common medical syringe can be used to directly inject thecomposition into a subcutaneous tumor. Alternatively, the compositioncan be topically applied to the tumor by bathing the tumor in thedisclosed liquid composition. Likewise, the tumor can be perfused withthe disclosed composition over a period of time using any suitabledelivery device, e.g., a catheter. Other routes of administration can beused to deliver the composition to the human. Some routes can provide amore immediate and more effective reaction than other routes. In someembodiments, the composition is administered to a surface of the subjectselected from the group of dermal and mucosal surfaces and combinationsthereof. For example, the disclosed compositions can be applied orinstilled into body cavities, absorbed through the skin, inhaled, oradministered parenterally via, for instance, intramuscular orintraarterial administration. In some embodiments, the disclosedcompositions parenterally administered to a human are specificallytargeted to particular cells, e.g., cancer cells.

Methods of Formulating

This disclosure is also directed to methods of formulating, comprisingproviding an ADC comprising a benzodiazepine in an aqueous solution,adding to the aqueous solution comprising the ADC a small hydrophobicmolecule selected from the group consisting of betaines and amino acidswith hydrophobic side chains. In some embodiments, the method furthercomprises adjusting the pH of the aqueous solution to between about 4.0and about 4.5. In some embodiments, the method further compriseslyophilizing the solution. In some embodiments, the method furthercomprises reconstituting the lyophilized composition.

In further embodiments of the method, the addition of a smallhydrophobic molecule reduces RSA in the aqueous solution by about 30% toabout 40%. In some embodiments, the addition of a small hydrophobicmolecule reduces RSA in the aqueous solution by about 40% to about 50%.In certain embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 50% to about 60%. Infurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 60% to about 70%. In stillfurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 70% to about 80%. In yetfurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the aqueous solution by about 80% to about 90%. In someembodiments, the addition of a small hydrophobic molecule reduces RSA inthe aqueous solution by about 90% to 100%. In still further embodiments,the addition of a small hydrophobic molecule eliminates RSA in theaqueous solution. In some embodiments, the amount of RSA is measured bymultiangle light scattering. In some further embodiments, the amount ofRSA is measured by dynamic light scattering.

In some embodiments, the method further comprises lyophilizing theaqueous solution, thereby obtaining a lyophilized composition. Incertain embodiments, the method further comprises reconstituting thelyophilized composition, thereby creating a reconstituted lyophilizedcomposition. In further embodiments of the method, the addition of asmall hydrophobic molecule reduces RSA in the reconstituted lyophilizedcomposition by about 30% to about 40%. In some embodiments, the additionof a small hydrophobic molecule reduces RSA in the reconstitutedlyophilized composition by about 40% to about 50%. In certainembodiments, the addition of a small hydrophobic molecule reduces RSA inthe reconstituted lyophilized composition by about 50% to about 60%. Infurther embodiments, the addition of a small hydrophobic moleculereduces RSA in the reconstituted lyophilized composition by about 60% toabout 70%. In still further embodiments, the addition of a smallhydrophobic molecule reduces RSA in the reconstituted lyophilizedcomposition by about 60% to about 70%. In yet further embodiments, theaddition of a small hydrophobic molecule reduces RSA in thereconstituted lyophilized composition by about 70% to about 80%. In someembodiments, the addition of a small hydrophobic molecule reduces RSA inthe reconstituted lyophilized composition by about 80% to about 90%. Incertain embodiments, the addition of a small hydrophobic moleculereduces RSA in the reconstituted lyophilized composition by about 90% to100%. In some embodiments, the addition of a small hydrophobic moleculeeliminates RSA in the reconstituted lyophilized composition.

The descriptions of the ADCs comprising a benzodiazepine, the smallhydrophobic molecules, excipients (e.g., buffering agents, surfactants,sugars, etc.), and other components described herein are also applicableto the compositions that are used in the methods of treating.

Methods of Reducing RSA

This disclosure is also directed to methods of reducing RSA in ADCscomprising benzodiazepines. One aspect is directed to methods ofreducing RSA in an ADC comprising a benzodiazepine, the methodcomprising providing an ADC comprising a benzodiazepine in an aqueoussolution, wherein the ADC exhibits RSA, adding to the aqueous solution asmall hydrophobic molecule selected from the group consisting ofbetaines and amino acids with hydrophobic side chains, wherein the smallhydrophobic molecule reduces or inhibits RSA. In some embodiments, themethod further comprises detecting reversible self-association. Incertain embodiments, the method further comprises lyophilizing theaqueous solution. In further embodiments, the method further comprisesreconstituting a lyophilized composition.

In some embodiments, RSA is eliminated in the disclosed compositions. Insome embodiments, RSA is decreased by about 90% to 100% in the disclosedcompositions. In certain embodiments, RSA is decreased by about 80% toabout 90% in the disclosed compositions. In some embodiments, RSA isdecreased by about 70% to about 80% in the disclosed compositions. Infurther embodiments, RSA is decreased by about 60% to about 70% in thedisclosed compositions. In still further embodiments, the RSA isdecreased by about 50% to about 60% in the disclosed compositions. Inyet further embodiments, RSA is decreased by about 40% to about 50% inthe disclosed compositions. In some embodiments, RSA is decreased byabout 30% to about 40% in the disclosed compositions. In someembodiments, the method further comprises adjusting the pH of thesolution to between about 4.0 to about 4.5.

The descriptions of the ADCs comprising benzodiazepine, the smallhydrophobic molecules, excipients (e.g., buffering agents, surfactants,sugars, etc.), and other components described herein are also applicableto the compositions that are used in the methods of reducing RSA.

EXAMPLES Example 1: Examination of RSA

This example demonstrates the use of dynamic light scattering andsedimentation velocity analytical ultracentrifugation as techniques forevaluating the extent of reversible self-association in anindolinobenzodiazepine ADC, huMy9-6-DGN462.

Dynamic Light Scattering measures the time-dependent fluctuation in theintensity of light scattered from the proteins or antibodies in solutionat a fixed scattering angle. As the protein or antibody or ADC moleculesundergo Brownian motion, their relative positions change with time.Small molecules, which diffuse quickly, generate signals that fluctuaterapidly, while larger proteins and antibodies generate slower signalfluctuations. The translational diffusion coefficient, D_(t), is relatedto the intensity autocorrelation function of the time-dependentfluctuation in intensity. The hydrodynamic diameter can be determinedusing the Stokes-Einstein relation [d_(h)=K_(T)/3πηD_(t), where d_(h) isthe hydrodynamic diameter, K_(T) is the Boltzmann constant, η isviscosity, and D_(t) is the translational diffusion coefficient].Scattering intensity data are processed using DLS instrument software todetermine the value for the translational diffusion coefficient and thesize distribution of the scattering molecules, i.e., the protein orantibody specimen.

All proteins will aggregate to some extent during quiescent storage asthe result of exposure of hydrophobic patches from partial unfoldingthat occurs with fluctuations between the native and non-native states.These aggregates do not dissociate with changes in pH or dilution, butrequire the introduction of chaotropes such as guanidine or urea todissociate. When examined by dynamic light scattering techniquessolutions that contain small amounts of aggregated protein, do not looksubstantially different than a solution of pure monomeric protein, i.e.,the hydrodynamic diameter and diffusion coefficients remain relativelyconstant regardless of solution characteristics such as concentration.

However, in the case of reversible self-association, where a change inthe solution properties, such as dilution, can effect a change in theassociation state (i.e., disrupt the self-association), DLS can be usedto measure a unique diffusion coefficient for a given concentration. Aplot of translational diffusion coefficient against protein or antibodyconcentration yields a best fit line with slope m and a y-intercept, b.A line where the slope m, is positive indicates a net repulsiveinteraction of the proteins or antibodies, while a negative slope isindicative of a net attractive interaction. This can be seen in FIG. 1.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) measuresthe rate at which molecules in solution move in response to centrifugalforce generated in a centrifuge. In SV-AUC the sample is spun at a veryhigh speed (42-60 k rpm) and the evolution of the concentration gradientis monitored by UV absorbance optics. The high centrifugal force rapidlydepletes the protein or antibody from the center of the rotor and formsa boundary that moves towards the outside of the rotor over time. Therate that this boundary moves is a measure of the sedimentationcoefficient and is related to the molecular weight and molecular shape,generally represented by the equation s=m/6πηr₀ where m is molecularweight, η is viscosity, and r₀ is the radius of the particle. From thesedata a distribution of the variously sized components in the sample canbe measured.

The rate at which the boundary moves is also dependent on the diffusionand frictional forces that act in the opposite direction ofsedimentation of the molecule. The minimum width of the sedimentationboundary is related to the diffusion coefficient. The presence ofseveral species with similar sedimentation coefficients will cause theboundary to be broader than expected.

In the case of reversibly self-associating molecules, the sedimentationboundary is broader than expected due to the presence of higher orderedoligomers that are stable over the time scale of sedimentation. Thismanifests as diffusion that is much faster than would be expected formolecules of the measured sedimentation coefficients. To account forthis, the shape of the molecule, which is inferred from the frictionalratio f/f₀, where f is the frictional coefficient for the protein orantibody and f₀ is the frictional coefficient for a hard solid sphere ofradius r, is calculated to be more spherical. For reversiblyself-associating antibodies which are more elongated than globular, thefrictional ratio is considerably smaller (˜1) than for non-associatingantibodies (˜1.5).

In the case of reversibly self-associating molecules, there is also aconcentration dependent measure of the distribution of the components inthe sample. In SV-AUC, when a set of serially diluted samples is run,the relative proportions of the components change as well as themeasured sedimentation coefficient. As the solution becomes more dilute,the sedimentation coefficient begins to approach the expected value. Inthe case of antibodies this is ˜6.5 s. This can be seen in FIGS. 2A &2B.

Example 2a: Influence of Drug Load on RSA

This example demonstrates the impact of drug load (DAR) on the extent ofreversible self-association in an indolinobenzodiazepine ADC. DARrepresents an average of indolinobenzodiazepine molecules attached tothe antibodies.

Conjugates comprising the huMy-9-6 monoclonal antibody chemicallycoupled to the indolinobenzodiazepine DGN462 via a4-(2-pyridinyldithio)-2-sulfo-,1-(2,5-dioxo-1-pyrrolidinyl) butanoicacid ester (sSPDB) linker (the ADC is referred to as“huMy9-6-sSPDB-DGN462”) were prepared using methods described herein andknown in the art (see, e.g., U.S. Pat. No. 8,889,669, which is hereinincorporated by reference) to yield drug to antibody ratios (DAR) of1.8, 2.4, and 2.8. huMy9-6-sSPDB-DGN462 conjugates with differing drugloads, but the same antibody concentration (about 2 mg/mL), wereformulated in 20 mM histidine, 8% trehalose, 0.02% polysorbate 20, pH6.1.

As can be seen in FIG. 3, compositions with a lower drug load hadsmaller hydrodynamic diameters than those with higher drug loadssuggesting that the intermolecular interactions are between theindolinobenzodiazepine moieties attached to each antibody.

Example 2b: Reduced RSA Compositions

This example demonstrates the production of a composition that reducesor inhibits reversible self-association comprising an ADC comprising anantibody chemically coupled to an indolinobenzodiazepine (DGN462),buffering agent, surfactant, hydrophobic amino acid, sugar, and water.

A conjugate comprising the huMy-9-6 monoclonal antibody chemicallycoupled to the indolinobenzodiazepine DGN462 via a4-(2-pyridinyldithio)-2-sulfo-,1-(2,5-dioxo-1-pyrrolidinyl) butanoicacid ester (sSPDB) linker (the ADC is referred to as“huMy9-6-sSPDB-DGN462”) was prepared using methods described herein andknown in the art (see, e.g., U.S. Pat. No. 8,889,669).huMy9-6-sSPDB-DGN462 conjugates were formulated as follows: (a) 0.2mg/mL ADC, 20 mM histidine, 8% trehalose, 0.02% polysorbate 20, pH 6.1;(b) 0.2 mg/mL ADC, 10 mM succinate, 8% trehalose, pH 4.2; (c) 0.5 mg/mLADC, 10 mM sodium succinate, 280 mM betaine, pH 4.2; and (d) 0.5 mg/mLADC, 10 mM sodium succinate, 280 mM proline, pH 4.2. The results ofanalysis of dynamic light scattering demonstrating the effects of theformulation pH and excipients on reversible self-association are setforth in FIG. 4. As can be seen in FIG. 4, the formulations with prolineand betaine have the least negative slope, indicating a lower amount ofnet attractive interaction when compared to the other formulations. Theresults of SV-AUC for the 10 mM succinate, 280 mM proline formulationand the 10 mM succinate, 280 mM betaine formulation are set forth inFIG. 5. The results show that succinate/proline or succinate/betaineformulations are superior to trehalose/histidine pH 6.1 formulations forreducing RSA.

These experiments demonstrate the surprisingly reduced RSA in thecompositions of this disclosure.

Example 3a: Reduced RSA Formulation: AbX-D2 Conjugate with Proline

This example demonstrates the production of a composition for reducingor inhibiting reversible self-association comprising a conjugatecomprising an antibody chemically coupled to an indolinobenzodiazepineD2, buffering agent, surfactant, hydrophobic amino acid, sugar, andwater.

A conjugate comprising a monoclonal antibody AbX chemically coupled tothe indolinobenzodiazepine D2 (herein referred to as “AbX-D2”) isprepared using methods described herein and in U.S. ApplicationPublication No. US20160082114A1, which is herein incorporated byreference in its entirety. Compositions comprising the AbX-D2 conjugateare formulated as follows: (a) 20 mM histidine, 8% trehalose, 0.02%polysorbate 20, pH 6.1; (b) 10 mM acetate, 8% trehalose, pH 4.2; (c) 10mM sodium succinate, 280 mM proline or 280 mM Betaine, pH 4.2; and (d)10 mM sodium succinate, 8% trehalose, and optionally 0.02% polysorbate,pH4.2. For each of the compositions, 2-200 μM bisulfate may also beincluded.

Example 3b: Reduced RSA Formulation: huMov19-sSPDB-D1 Conjugate withLeucine

This example demonstrates the production of a composition for reducingor inhibiting reversible self-association comprising a conjugatecomprising an antibody chemically coupled to an indolinobenzodiazepineD3, buffering agent, surfactant, hydrophobic amino acid, sugar, andwater.

A conjugate comprising the huMov19 monoclonal antibody chemicallycoupled to the indolinobenzodiazepine D1 (herein referred to as“huMov19-sSPDB-D1”) is prepared using methods described herein (see,e.g., U.S. Application Publication No. US20160106863). Compositionscomprising the huMov19-sSPDB-D1 conjugate are formulated as follows: (a)20 mM histidine, 8% trehalose, 0.02% polysorbate 20, pH 6.1; (b) 10 mMacetate, 8% trehalose, pH 4.2; (c) 10 mM sodium succinate, 125 mMleucine, pH 4.2; and (d) 10 mM sodium succinate, 8% trehalose, andoptionally 0.02% polysorbate, pH4.2.

Example 3c: Reduced RSA Formulation: huMov19-sSPDB-D4 Conjugate withIsoleucine

This example demonstrates the production of a composition for reducingor eliminating reversible self-association comprising a conjugatecomprising an antibody chemically coupled to an indolinobenzodiazepineD4, buffering agent, surfactant, hydrophobic amino acid, sugar, andwater.

A conjugate comprising the huMov19 monoclonal antibody chemicallycoupled to the indolinobenzodiazepine D4 (herein referred to as“huMov19-sSPDB-D4”) is prepared using methods described herein (see,e.g., U.S. Application Publication No: US20160082114A1). Compositionscomprising the huMov19-sSPDB-D4 conjugate are formulated as follows: (a)20 mM histidine, 8% trehalose, 0.02% polysorbate 20, pH 6.1; (b) 10 mMacetate, 8% trehalose, pH 4.2; (c) 10 mM sodium succinate, 125 mMisoleucine, pH 4.2; and (d) 10 mM sodium succinate, 8% trehalose, andoptionally 0.02% polysorbate, pH4.2.

Example 4: Methods of Making D1

Compound 1a:

To a stirred solution of (5-amino-1,3-phenylene)dimethanol (1.01 g, 6.59mmol) in anhydrous dimethylformamide (16.48 mL) and anhydroustetrahydrofuran (16.48 ml) was added4-methyl-4-(methyldisulfanyl)pentanoic acid (1.281 g, 6.59 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (2.53 g,13.19 mmol), and 4-dimethylaminopyridine (0.081 g, 0.659 mmol). Theresulting mixture was stirred for 18 hours at room temperature. Thereaction was quenched with saturated ammonium chloride solution andextracted with ethyl acetate (3×50 mL). The organic extracts were washedwith water and brine, then dried over anhydrous sodium sulfate. Thesolution was filtered and concentrated in vacuo and the resultingresidue was purified by silica gel chromatography (Ethylacetate/Hexanes) to obtain compound 1a as a white solid (0.70 g, 32%yield). ¹H NMR (400 MHz, DMSO-d6: δ 9.90 (s, 1H), 7.43 (s, 2H), 6.93 (s,1H), 5.16 (t, 2H, J=5.7 Hz), 4.44 (d, 4H, J=5.7 Hz), 2.43 (s, 3H),2.41-2.38 (m, 2H), 1.92-1.88 (m, 2H), 1.29 (s, 6H). MS (m/z), found330.0 (M+1)⁺.

Compound 1b:

To a cooled (−10° C.) solution of compound 1a (219 mg, 0.665 mmol) inanhydrous dichloromethane (6.65 mL) was added triethylamine (463 μl,3.32 mmol) followed by dropwise addition of methanesulfonic anhydride(298 mg, 1.662 mmol). The mixture stirred at −10° C. for 2 hours, thenthe mixture was quenched with ice water and extracted with cold ethylacetate (2×30 mL). The organic extracts were washed with ice water,dried with anhydrous sodium sulfate, filtered and concentrated underreduced pressure to obtain the crude dimesylate.

The crude dimesylate (227 mg, 0.467 mmol) and IGN monomer A (303 mg,1.028 mmol) were dissolved in anhydrous DMF (3.11 mL). Potassiumcarbonate (161 mg, 1.169 mmol) was added and the mixture stirred for 18hours at room temperature. Deionized water was added and the resultingprecipitate was filtered and rinsed with water. The solid wasre-dissolved in dichloromethane and washed with water. The organic layerwas dried with anhydrous magnesium sulfate, filtered, and concentrated.The crude residue was purified by silica gel chromatography(Methanol/Dichloromethane) to give compound 1b (227 mg, 36% yield). MS(m/z), found 882.5 (M+1)⁺.

Compound 1c:

To a suspension of compound 1b (227 mg, 0.167 mmol) in anhydrous1,2-dichloroethane (3.346 mL) was added sodium triacetoxyborohydride(STAB) (37.3 mg, 0.167 mmol). The mixture was stirred at room temp forone hour upon which it was quenched with saturated ammonium chloridesolution. The mixture was extracted with dichloromethane and washed withbrine. The organic layer was dried with anhydrous magnesium sulfate,filtered and concentrated. The crude residue was purified by RP-HPLC(C18, Water/Acetonitrile). Fractions containing desired product wereextracted with dichloromethane, dried with anhydrous magnesium sulfate,filtered and concentrated to give compound 1c (35 mg, 19% yield). MS(m/z), found 884.3 (M+1)⁺.

Compound 1d:

To a solution of compound 1c (18 mg, 0.017 mmol) in acetonitrile (921μL) and methanol (658 μL) was added tris(2-carboxyethyl)phosphinehydrochloride (TCEP) (17.51 mg, 0.060 mmol) (neutralized with saturatedsodium bicarbonate solution (0.2 mL) in sodium phosphate buffer (132 μL,0.75 M, pH 6.5). The mixture was stirred at room temperature for 3.5hours, then diluted with dichloromethane and deionized water. Theorganic layer was separated, washed with brine, dried with anhydroussodium sulfate, filtered and concentrated under reduced pressure toobtain the crude thiol. MS (m/z), found 838.3 (M+1)⁺.

The crude thiol from step 5 (15.5 mg, 0.018 mmol) was dissolved in2-propanol (1.23 mL). Deionized water (617 μL) and sodium bisulfate(5.77 mg, 0.055 mmol) were added and the mixture stirred for five hoursat room temperature. The reaction was frozen in an acetone/dry ice bath,lyophilized, and purified by RP-HPLC (C18, deionizedwater/acetonitrile). Fractions containing desired product were frozenand lyophilized to give compound(12S,12aS)-9-((3-(4-mercapto-4-methylpentanamido)-5-((((R)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)benzyl)oxy)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-12-sulfonicacid (compound 1d, also referred to herein as D1) (6.6 mg, 39% yield).MS (m/z), found 918.2 (M−1)⁻.

Example 5: Preparation of huMOV19-Sulfo-SPDB-1d

A reaction containing 2.0 mg/mL huMOV19 antibody and 6 molar equivalentsof sulfo-SPDB-1d in situ mixture by linker in 50 mM HEPES(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and15% v/v DMA (N,N-Dimethylacetamide) cosolvent was allowed to conjugatefor 6 hours at 25° C. The in situ mixture was prepared by reacting 1.5mM sulfo-SPDB linker with 1.95 mM of compound 1d in 100% DMA for 4 hoursin the presence of 10 mM N,N-Diisopropylethyl amine (DIPEA). Free thiolwas then capped by adding a 3-fold excess of maleimido-propionic acid.

Post-reaction, the conjugate was purified and buffer exchanged into 100mM Arginine, 20 mM Histidine, 2% sucrose, 0.01% Tween-20, 50 μM sodiumbisulfite formulation buffer pH 6.1 using NAP desalting columns(Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis wasperformed in the same buffer for 20 hours at 4° C. utilizingSlide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO).

The purified conjugate was found to have an average of 2.5 molecules ofcompound 1d linked per antibody (by UV-Vis using molar extinctioncoefficients ε_(330 nm)=15,280 cm⁻¹M⁻¹ and ε_(280 nm)=30, 115 cm⁻¹M⁻¹for compound 1d, and ε_(280 nm)=201,400 cm⁻¹M⁻¹ for huMOV19 antibody),95% monomer (by size exclusion chromatography), <0.1% unconjugatedcompound 1d (by acetone precipitation, reverse-phase HPLC analysis) anda final protein concentration of 1.8 mg/ml. The conjugated antibody wasfound to be >80% intact by gel chip analysis.

Example 6: Methods of Making D2

Synthesis of 2, 5-dioxopyrrolidin-1-yl6-(((S)-1-(((S)-1-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((R)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexanoate,compound 90, also referred to herein as D2.

Step 1:

(S)-2-(((benzyloxy)carbonyl)amino)propanoic acid (5 g, 22.40 mmol) and(S)-tert-butyl 2-aminopropanoate hydrochloride (4.48 g, 24.64 mmol) weredissolved in anhydrous DMF (44.8 mL). EDC.HCl (4.72 g, 24.64 mmol), HOBt(3.43 g, 22.40 mmol), and DIPEA (9.75 mL, 56.0 mmol) were added. Thereaction stirred under argon, at room temperature, overnight. Thereaction mixture was diluted with dichloromethane and then washed withsaturated ammonium chloride, saturated sodium bicarbonate, water, andbrine. The organic layer was dried over sodium sulfate and concentrated.The crude oil was purified via silica gel chromatography (Hexanes/EthylAcetate) to yield compound 81 (6.7 g, 85% yield). ¹H NMR (400 MHz,CDCl₃): δ 7.38-7.31 (m, 5H), 6.53-6.42 (m, 1H), 5.42-5.33 (m, 1H), 5.14(s, 2H), 4.48-4.41 (m, 1H), 4.32-4.20 (m, 1H), 1.49 (s, 9H), 1.42 (d,3H, J=6.8 Hz), 1.38 (d, 3H, J=7.2 Hz).

Step 2:

Compound 81 (6.7 g, 19.12 mmol) was dissolved in methanol (60.7 mL) andwater (3.03 mL). The solution was purged with argon for five minutes.Palladium on carbon (wet, 10%) (1.017 g, 0.956 mmol) was added slowly.The reaction was stirred overnight under an atmosphere of hydrogen. Thesolution was filtered through Celite, rinsed with methanol andconcentrated. It was azeotroped with methanol and acetonitrile and theresulting oil was placed directly on the high vacuum to give compound 82(4.02 g, 97% yield) which was used directly in the next step. ¹H NMR(400 MHz, CDCl₃): δ 7.78-7.63 (m, 1H), 4.49-4.42 (m, 1H), 3.55-3.50 (m,1H), 1.73 (s, 2H), 1.48 (s, 9H), 1.39 (d, 3H, J=7.2 Hz), 1.36 (d, 3H,J=6.8 Hz).

Step 3:

Compound 82 (4.02 g, 18.59 mmol) and mono methyladipate (3.03 mL, 20.45mmol) were dissolved in anhydrous DMF (62.0 mL). EDC.HCl (3.92 g, 20.45mmol), HOBt (2.85 g, 18.59 mmol) and DIPEA (6.49 mL, 37.2 mmol) wereadded. The mixture was stirred overnight at room temperature. Thereaction was diluted with dichloromethane/methanol (150 mL, 5:1) andwashed with saturated ammonium chloride, saturated sodium bicarbonate,and brine. It was dried over sodium sulfate, filtered and stripped. Thecompound was azeotroped with acetonitrile (5×), then pumped on the highvacuum at 35° C. to give compound 83 (6.66 g, 100% yield). The crudematerial was taken onto next step without purification. ¹H NMR (400 MHz,CDCl₃): δ 6.75 (d, 1H, J=6.8 Hz), 6.44 (d, 1H, J=6.8 Hz), 4.52-4.44 (m,1H), 4.43-4.36 (m, 1H), 3.65 (s, 3H), 2.35-2.29 (m, 2H), 2.25-2.18 (m,2H), 1.71-1.60 (m, 4H), 1.45 (s, 9H), 1.36 (t, 6H, J=6.0 Hz).

Step 4:

Compound 83 (5.91 g, 16.5 mmol) was stirred in TFA (28.6 mL, 372 mmol)and deionized water (1.5 mL) at room temperature for three hours. Thereaction mixture was concentrated with acetonitrile and placed on highvacuum to give crude compound 84 as a sticky solid (5.88 g, 100% yield).¹H NMR (400 MHz, CDCl₃): δ 7.21 (d, 1H, J=6.8 Hz), 6.81 (d, 1H, J=7.6Hz), 4.69-4.60 (m, 1H), 4.59-4.51 (m, 1H), 3.69 (s, 3H), 2.40-2.33 (m,2H), 2.31-2.24 (m, 2H), 1.72-1.63 (m, 4H), 1.51-1.45 (m, 3H), 1.42-1.37(m, 3H).

Step 5:

Compound 84 (5.6 g, 18.52 mmol) was dissolved in anhydrousdichloromethane (118 mL) and anhydrous methanol (58.8 mL).(5-amino-1,3-phenylene)dimethanol (2.70 g, 17.64 mmol) and EEDQ (8.72 g,35.3 mmol) were added and the reaction was stirred at room temperature,overnight. The solvent was stripped and ethyl acetate was added. Theresulting slurry was filtered, washed with ethyl acetate and dried undervacuum/N₂ to give compound 85 (2.79 g, 36% yield). ¹H NMR (400 MHz,DMSO-d6): δ 9.82 (s, 1H), 8.05, (d, 1H, J=9.2 Hz), 8.01 (d, 1H, J=7.2Hz), 7.46 (s, 2H), 6.95 (3, 1H), 5.21-5.12 (m, 2H), 4.47-4.42 (m, 4H),4.40-4.33 (m, 1H), 4.33-4.24 (m, 1H), 3.58 (s, 3H), 2.33-2.26 (m, 2H),2.16-2.09 (m, 2H), 1.54-1.46 (m, 4H), 1.30 (d, 3H, J=7.2 Hz), 1.22 (d,3H, J=4.4 Hz).

Step 6:

Compound 85 (0.52 g, 1.189 mmol) and carbon tetrabromide (1.183 g, 3.57mmol) were dissolved in anhydrous DMF (11.89 mL). Triphenylphosphine(PPH3) (0.935 g, 3.57 mmol) was added and the reaction stirred underargon for four hours. The reaction mixture was diluted with DCM/MeOH(10:1) and washed with water and brine, dried over sodium sulfate,filtered, and concentrated. The crude material was purified by silicagel chromatography (DCM/MeOH) to give compound 86 (262 mg, 39% yield).¹H NMR (400 MHz, DMSO-d6): δ 10.01 (s, 1H), 8.11 (d, 1H, J=6.8 Hz), 8.03(d, 1H, J=6.8 Hz), 7.67 (s, 2H), 7.21 (s, 1H), 4.70-4.64 (m, 4H),4.40-4.32 (m, 1H), 4.31-4.23 (m, 1H), 3.58 (s, 3H), 2.34-2.26 (m, 2H),2.18-2.10 (m, 2H), 1.55-1.45 (m, 4H), 1.31 (d, 3H, J=7.2 Hz), 1.21 (d,3H, J=7.2 Hz).

Step 7:

Dibromide compound 86 and IGN monomer compound 10 were dissolved in DMF(1.84 mL). Potassium carbonate was added and was stirred at roomtemperature overnight. Water was added to the reaction mixture toprecipitate the product. The slurry was stirred at room temperature andwas then filtered and dried under vacuum/N₂. The crude material waspurified by silica gel chromatography (dichloromethane/methanol) to givecompound 87 (336 mg, 74% yield). LCMS=5.91 min (15 min method). MS(m/z): 990.6 (M+1)⁺.

Step 8:

Diimine compound 87 was dissolved in 1,2-dichloroethane. NaBH(OAc)₃(STAB) was added to the reaction mixture and was stirred at roomtemperature for 1 h. The reaction was diluted with CH₂Cl₂ and wasquenched with sat'd aq NH₄Cl solution. The layers were separated and waswashed with brine, dried over Na₂SO₄ and concentrated. The crudematerial was purified via RPHPLC (C18 column, Acetonitrile/Water) togive compound 88 (85.5 mg, 25% yield). LCMS=6.64 min (15 min method). MS(m/z): 992.6 (M+1)⁺.

Step 9:

Methylester compound 88 was dissolved in 1,2-dichloroethane.Trimethylstannanol was added to the reaction mixture and was heated at80° C. overnight. The reaction mixture was cooled to room temperatureand was diluted with water. The aqueous layer was acidified to pH ˜4with 1 M HCl. The mixture was extracted with CH₂Cl₂/MeOH (10:1, 3×20mL). The combined organic layers were washed with brine and was driedover Na₂SO₄ and concentrated. The crude material was passed through asilica plug to give compound 89 (48.8 mg, 80% yield). LCMS=5.89 min (15min method). MS (m/z): 978.6 (M+1)⁺.

Step 10:

EDC.HCl was added to a stirred solution of acid compound 89 andN-hydroxysuccinamide in CH₂Cl₂ at rt. The reaction mixture was stirredfor 2 h. The reaction mixture was diluted with CH₂Cl₂ and was washedwith water and brine. The organic layer was dried over Na₂SO₄, filteredand concentrated. The crude material was purified via RPHPLC (C18column, Acetonitrile/Water) to give 2,5-dioxopyrrolidin-1-yl6-(((S)-1-(((S)-1-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((R)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexanoate,compound 90 (8.2 mg, 30% yield). LCMS=6.64 min (15 min method). MS(m/z): 1075.4 (M+1)⁺.

Example 7: Preparation of huMOV19-90

A reaction containing 2.0 mg/mL huMOV19 antibody and 3.9 molarequivalents of compound 90 (pretreated with 5-fold excess of sodiumbisulfate in 95:5 DMA:50 mM succinate pH 5.5 for 4 hours at 25° C.) in15 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5buffer and 15% v/v DMA (N,N-Dimethylacetamide) cosolvent was incubatedfor 4 hours at 25° C. Post-reaction, the conjugate was purified andbuffer exchanged into 10 mM succinate, 50 mM sodium chloride, 8.5% w/vsucrose, 0.01% Tween-20, 50 μM sodium bisulfite pH 6.2 formulationbuffer using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GEHealthcare). Dialysis was performed in the same buffer for 4 hours atroom temperature and then overnight at 4° C. utilizing Slide-a-Lyzerdialysis cassettes (ThermoScientific 30,000 MWCO).

The purified conjugate was found to have a final protein concentrationof 1.8 mg/ml and an average of 2.7 molecules of compound 90 linked perantibody (by UV-Vis using molar extinction coefficientsε_(330 nm)=15,280 cm⁻¹M⁻¹ and ε_(280 nm)=30, 115 cm⁻¹M⁻¹ for compound90, and ε_(280 nm)=201,400 cm⁻¹M⁻¹ for huMOV19 antibody); 98.3% monomer(by size exclusion chromatography); and <1.1% unconjugated compound 90(by acetone precipitation, reverse-phase HPLC analysis). The MSspectrometry data is shown in FIG. 6. DAR0 represents an unconjugatedantibody, i.e., an antibody that has no benzodiazepines conjugated toit. DAR6 represents an antibody with six benzodiazepines linked to it.The peaks in the middle correspond, from left to right, DAR1, DAR2,DAR3, DAR4, and DAR5.

Example 8. Synthesis of Compound 107, Also Referred to Herein as D4

Step 1:

Compound 82 (500 mg, 2.31 mmol), 4-methyl-4-(methyldisulfanyl)pentanoicacid (449 mg, 2.31 mmol), EDC.HCl (465 mg, 2.43 mmol), HOBt (354 mg,2.31 mmol), and DIPEA (0.81 mL, 4.62 mmol) were dissolved in DMF (7.7mL) and stirred overnight until the reaction was complete. The reactionwas diluted with ethyl acetate and washed with saturated sodiumbicarbonate, saturated ammonium chloride, and twice with water. Theorganic was dried and concentrated in vacuo to give compound 100 (875mg, 96% yield) which was used directly in the next step. ¹H NMR (400MHz, DMSO): δ 8.15 (d, 1H, J=6.8 Hz), 8.02 (d, 1H, J=6.8 Hz), 4.26-4.33(m, 1H), 4.03-4.12 (m, 1H), 2.41 (s, 3H), 2.18-2.22 (m, 2H), 1.76-1.80(m, 2H), 1.39 (s, 9H), 1.24 (s, 6H), 1.24 (d, 3H, J=7.2 Hz), 1.19 (d,3H, J=7.2 Hz).

Step 2:

TFA (2.6 ml) and water (0.17 ml) were added to neat Compound 100 (875mg, 2.23 mmol) and were stirred at room temperature until the reactionwas complete. The reaction was diluted and azeotroped with acetonitrileto obtain a sticky oil. It was then diluted with acetonitrile and water,frozen and lyophilized to give compound 101 (1 g, 100% yield) as an offwhite solid that was used without further purification. LCMS=3.99 min (8min method). MS (m/z): 337.0 (M+1)⁺.

Step 3:

Compound 101 (923 mg, 1.65 mmol) and (5-amino-1,3-phenylene)dimethanol(240 mg, 1.57 mmol) were dissolved in DMF (5.2 ml). EDC.HCl (601 mg,3.13 mmol), and DMAP (96 mg, 0.78 mmol) were added at room temperatureand the reaction was stirred overnight at room temperature. The reactionwas diluted with ethyl acetate and washed with water three times. Theorganic layer was dried, concentrated in vacuo and purified by silicagel chromatography (DCM/MeOH) to give Compound 102 (150 mg, 20% yield).LCMS=3.91 min (8 min method). MS (m/z): 472.2 (M+1)⁺. ¹H NMR (400 MHz,MeOD): δ 9.69 (s, 1H), 8.21 (d, 1H, J=6.8 Hz), 8.18 (d, 1H, J=6.8 Hz),7.52 (s, 2H), 7.12 (s, 1H), 4.58 (s, 4H), 4.44-4.48 (m, 1H), 4.29-4.32(m, 1H), 3.34 (s, 2H), 2.38 (s, 3H), 2.34-2.40 (m, 2H), 1.90-1.95 (m,2H), 1.43 (d, 3H, J=7.2 Hz), 1.36 (d, 3H, J=7.2 Hz), 1.30 (s, 6H).

Step 4:

Compound 102 was suspended in anhydrous DCM. Anhydrous DMF was addeduntil the solution became homogeneous. The solution was cooled to −10°C. in an acetone/dry ice bath. Triethylamine was added, followed bymethanesulfonic anhydride. The mixture stirred at −10° C. for 1 hour.The reaction was quenched with ice water and extracted with cold ethylacetate/methanol (20:1). The organic layer was washed with ice water anddried over anhydrous sodium sulfate, filtered and concentrated. Thecrude material was dried under high vacuumed to give Compound 103 (174mgs, 101% yield) that was used directly in the next step without furtherpurification. LCMS=4.95 min (8 min method).

Step 5:

Dimesylate compound 103 (435 mg, 1.11 mmol) was dissolved in DMF. IGNmonomer compound 10 was added, followed by and K₂CO₃ and was stirred atroom temperature under Ar overnight. Water was added to precipitate outthe product. The slurry was stirred for 5 min, filtered and dried undervacuum/N₂. The crude solid contained compound 104 (203 mg, 44% yield,60% purity) which was used without further purification. LCMS=5.68 min(8 min method). MS (m/z): 1024.3 (M+1)⁺.

Step 6:

Diimine compound 104 was dissolved in 1,2-dichloroethane. NaBH(OAc)₃ wasadded to the reaction mixture and was stirred at rt. The reaction wasdiluted with CH₂Cl₂ and was quenched with sat'd aq NH₄Cl solution (15mL). The layers were separated and was washed with brine, dried overNa₂SO₄ and concentrated. The crude residue was purified by RPHPLC (C18column, CH₃CN/H₂O, gradient, 50% to 65%) to yield mono imine compound105 as a solid (22 mg, 16% yield, 90% pure). LCMS=6.00 min (8 minmethod). MS (m/z): 1027.3 (M+1)⁺.

Step 7:

Compound 106 was dissolved in THF (0.5 mL) and ACN (0.23 mL) at roomtemperature. It was then prepared similarly to compound 98 in Example 9.The mixture was stirred until completion and then diluted with DCM andDI water. The organic layer was washed with brine, dried and filtered.The filtrate was concentrated to give the crude thiol, compound 106 (21mg, 100% yield) which was used directly in the next reaction. LCMS=5.67min (8 min method). MS (m/z): 980.4 (M+1)⁺.

Step 8:

Compound 106 (21 mg, 0.021 mmol) was suspended in 2-propanol (1428 μl)and water (714 μl). Sodium metabisulfite (22.30 mg, 0.214 mmol) wasadded and the reaction stirred at room temperature until completion. Thereaction mixture was diluted with acetonitrile/water, frozen andlyophilized. The resulting white powder was purified by RPHPLC (C18column, CH₃CN/H₂O, gradient, 20% to 40%) and the desired fractions werecollected and lyophilized to give compound 107 (5.3 mg, 23% yield).LCMS=5.67 min (8 min method). MS (m/z): 1060.2 (M−1)⁻.

Example 9. Preparation of huMOV19-Sulfo-SPDB-107 (or huMOV19-107)Conjugate

An in situ mix containing final concentrations of 1.95 mM Compound 107and 1.5 mM sulfo-SPDB Linker in succinate buffer (pH 5): DMA (30:70) wasincubated for 6 h before adding a 7-fold excess of 107-sulfo-SPDB-NHS toa reaction containing 4 mg/ml huMOV19 antibody in 15 mM HEPES pH 8.5(87:13, water: DMA). The solution was allowed to conjugate over night at25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 10mM Tris, 80 mM NaCl, 50 uM Bisulfite, 3.5% Sucrose, 0.01% Tween-20formulation buffer pH 7.6 using NAP desalting columns (Illustra SephadexG-25 DNA Grade, GE Healthcare). Dialysis was performed in the samebuffer over night at 4° C. utilizing Slide-a-Lyzer dialysis cassettes(ThermoScientific 10,000 MWCO).

The purified conjugate was found to have an average of 2.7 molecules ofcompound 107 linked per antibody (by UV/Vis and SEC using molarextinction coefficients ε_(330 nm)=15,484 cm⁻¹M⁻¹ and ε_(280 nm)=30, 115cm⁻¹M⁻¹ for compound 107, and ε_(280 nm)=201,400 cm⁻¹M⁻¹ for huMOV19antibody), 95% monomer (by size exclusion chromatography), and a finalprotein concentration of 1.1 mg/ml. The MS spectrometry data is shown inFIG. 7. DAR0 represents an unconjugated antibody, i.e., an antibody thathas no benzodiazepines conjugated to it. DAR5 represents an antibodywith five benzodiazepines linked to it. The peaks in the middlecorrespond, from left to right, DAR1, DAR2, DAR3, and DAR4.

Example 10. Reduced RSA with Low pH Succinate Buffers

This example demonstrates the production of compositions that reduce,inhibit, or eliminate reversible self-association where the compositionsinclude a conjugate comprising an antibody with an engineered cysteine(e.g., a non-naturally occurring cysteine introduced into the antibodyheavy chain or light chain in place of another non-cysteine amino acid)chemically coupled to an indolinobenzodiazepine, buffering agent,surfactant, sugar, and water.

Conjugates comprising the AbX monoclonal antibody chemically coupled tothe indolinobenzodiazepine D2(a) through engineered cysteines wereproduced. The conjugates were formulated as (a) 10 mM histidine, 8%trehalose, 0.01% polysorbate 20, pH 5.5; or (b) 10 mM sodium succinate,8% trehalose, 0.01% polysorbate 20, pH 4.2.

As shown in FIG. 8, the succinate and trehalose combination at pH 4.2(formula (b)) showed a greater reduction in reversible self-associationas measured by DLS when compared to the histidine trehalose combination(formula (a)).

These results demonstrate the ability of compositions disclosed hereinto reduce, inhibit, or eliminate reversible self-association at lower pHranges such as pH 4.2.

Example 11. Reduced RSA with Succinate Buffer

This example demonstrates the production of compositions that reduce,inhibit, or eliminate reversible self-association comprising a conjugatecomprising an antibody chemically coupled to an indolinobenzodiazepine,succinate-based buffering agent, surfactant, sugar, and water.

Conjugates comprising the huMy-9-6 monoclonal antibody chemicallycoupled to the indolinobenzodiazepine DGN462 via a4-(2-pyridinyldithio)-2-sulfo-,1-(2,5-dioxo-1-pyrrolidinyl) butanoicacid ester (sSPDB) linker (“huMy-9-6-sSPDB-DGN462”) were prepared usingmethods described herein and known in the art (see, e.g., U.S. Pat. No.6,441,163). The huMy-9-6-sSPDB-DGN462 conjugate was formulated asfollows: (a) 20 mM histidine, 8% trehalose, 0.02% polysorbate 20, pH6.1; (b) 10 mM acetate, 8% trehalose, pH 4.2; and (c) 10 mM sodiumsuccinate, 8% trehalose, pH 4.2.

The results of analysis by dynamic light scattering demonstrating theeffects of the formulation pH and buffering agent on reversibleself-association are set forth in FIG. 9. These results indicate thatsuccinate (formula (c)) as a buffering agent is more effective atreducing reversible self-association than acetate (formula (b)) in thepH range of 4.0 to 4.5, and both are more effective than histidine(formula (a)) at pH 6.1.

As also shown in FIG. 9, the succinate trehalose combination (formula(c)) is more effective than the acetate trehalose combination (formula(b)) at reducing reversible self-association at pH 4.2.

In addition, the data shown in FIG. 10 result from the assessment ofreversible self-association for the succinate-trehalose combination overthe range of pH4.2 to pH5.7. The results show that reduction inreversible self-association increases as the pH decreases, as shownherein with the use of a succinate buffer.

Example 12. Reduced RSA with Low pH Succinate Buffers

This example demonstrates the production of compositions that reduce,inhibit, or eliminate reversible self-association where the compositionsinclude a conjugate comprising an antibody with an engineered cysteine(e.g., a non-naturally occurring cysteine introduced into the antibodyheavy chain or light chain in place of another non-cysteine amino acid)chemically coupled to an indolinobenzodiazepine, buffering agent,surfactant, sugar, and water.

Conjugates comprising the AbX monoclonal antibody chemically coupled tothe indolinobenzodiazepine D2(a) through engineered cysteines wereproduced. The conjugate was formulated as 10 mM sodium succinate, 8%trehalose, 0.01% polysorbate 20, pH 4.0.

As shown in FIG. 11, the succinate and trehalose combination at pH 4.0showed an equivalent or greater reduction in reversible self-associationas measured by DLS when compared to the succinate/trehalose formulationat pH 4.2.

Using a site-specific conjugate, this example shows the reduction of RSAeven in a conjugate with a lower DAR. These results further demonstratethe ability of compositions disclosed herein to reduce, inhibit, oreliminate reversible self-association particularly at lower pH rangessuch as pH 4.0-4.5, even for site specific conjugates with a DAR of, forexample, 2.0.

EQUIVALENTS

It is to be understood that the foregoing description is intended toillustrate and not limit the scope of the invention, which is defined bythe scope of the appended claims. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific embodimentsdescribed specifically in this disclosure. Such equivalents, and otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of formulating an ADC, comprising (a) reducing reversibleself-association in a formulation by (i) providing an ADC comprising abenzodiazepine in an aqueous solution at a first pH, wherein the ADCexhibits reversible self-association; (ii) adjusting the pH of theaqueous solution to a second pH, wherein the second pH ranges from about4.0 to about 4.5, and wherein the adjustment of the pH from the first pHto the second pH reduces reversible self-association.
 2. The method ofclaim 1, wherein the second pH is about 4.2.
 3. The method of claim 1,wherein the benzodiazepine is selected from the group consisting of D1,D1(a), D2, D2(a), DGN462, DGN462(a), D3, D3(a), D4, D4(a), D5, D5(a),D6, and D6(a).
 4. The method of claim 1, wherein the ADC is selectedfrom the group consisting of Ab-sSPDB-D1, Ab-sSPDB-D1(a), Ab-D2,Ab-D2(a), Ab-sSPDB-DGN462, Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a),Ab-sSPDB-D4, Ab-sSPDB-D4(a), Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1,Ab-Ser-D1(a), Ab-Cys-DGN462, Ab-Cys-DGN462(a), Ab-Ser-DGN462,Ab-Ser-DGN462(a), Ab-Cys-D5, Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a).5. The method of claim 1, wherein the reversible self-association isreduced by about 70% to about 80%.
 6. The method of claim 1, wherein thereversible self-association is reduced by about 80% to about 90%.
 7. Themethod of claim 1, wherein the reversible self-association is reduced byabout 90% to 100%.
 8. The method of claim 1, further comprisinglyophilizing the aqueous solution, thereby creating a lyophilizedcomposition.
 9. The method of claim 8, further comprising reconstitutingthe lyophilized composition.
 10. The method of claim 1, wherein the ADCcomprises an antibody selected from the group consisting of huMy9-6,huB4, huDS6, huMov19, and huCD37-3.
 11. The method of claim 1, whereinthe ADC comprises a humanized CD123 antibody.
 12. The method of claim11, wherein the humanized CD123 antibody is AbX.
 13. The method of claim12, wherein the CD123 antibody is AbX₁.
 14. The method of claim 13,wherein AbX₁ comprises the heavy chain variable region CDR sequences SEQID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and the light chain variableregion CDR sequences SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:
 6. 15.The method of claim 13, wherein AbX₁ comprises a heavy chain variableregion domain at least about 90% identical to SEQ ID NO: 7 and a lightchain variable region at least about 90% identical to SEQ ID NO:
 9. 16.The method of claim 13, wherein AbX₁ comprises a heavy chain variableregion domain at least about 90% identical to SEQ ID NO: 11 and a lightchain variable region at least about 90% identical to SEQ ID NO:
 14. 17.The method of claim 13, wherein AbX1 comprises a heavy chain variableregion domain at least about 90% identical to SEQ ID NO: 12 and a lightchain variable region at least about 90% identical to SEQ ID NO:
 14. 18.The method of claim 12, wherein the CD123 antibody is AbX₂.
 19. Themethod of claim 18, wherein AbX₂ comprises a heavy chain variable regiondomain at least about 90% identical to SEQ ID NO: 8 and a light chainvariable region at least about 90% identical to SEQ ID NO:
 10. 20. Themethod of claim 18, wherein AbX2 comprises a heavy chain variable regiondomain at least about 90% identical to SEQ ID NO: 13 and a light chainvariable region at least about 90% identical to SEQ ID NO:
 15. 21. Themethod of claim 11, wherein the benzodiazepine is D5 or D5(a).
 22. Themethod of claim 1, further comprising adding trehalose dehydrate to theformulation.
 23. A composition comprising: (a) an ADC comprising abenzodiazepine; and (b) trehalose; wherein the composition has a pHranges from about 4.0 to about 4.5.
 24. The composition of 23, furthercomprising sodium succinate.
 25. The composition of 24, furthercomprising sodium bisulfate.
 26. The composition of any one of claims23-25, further comprising a surfactant.
 27. The composition of any oneof claims 23-26, wherein the benzodiazepine is selected from the groupconsisting of D1, D1(a), D2, D2(a), DGN462, DGN462(a), D3, D3(a), D4,D4(a), D5, D5(a), D6, and D6(a).
 28. The composition of any one ofclaims 23-27, wherein the ADC is selected from the group consisting ofAb-sSPDB-D1, Ab-sSPDB-D1(a), Ab-D2, Ab-D2(a), Ab-sSPDB-DGN462,Ab-sSPDB-DGN462(a), Ab-D3, Ab-D3(a), Ab-sSPDB-D4, Ab-sSPDB-D4(a),Ab-Cys-D1, Ab-Cys-D1(a), Ab-Ser-D1, Ab-Ser-D1(a), Ab-Cys-DGN462,Ab-Cys-DGN462(a), Ab-Ser-DGN462, Ab-Ser-DGN462(a), Ab-Cys-D5,Ab-Cys-D5(a), Ab-Ser-D6, and Ab-Ser-D6(a).
 29. The composition of anyone of claims 23-28, wherein the ADC comprises a humanized CD123antibody.
 30. The method of claim 29, wherein the humanized CD123antibody is AbX.
 31. The method of claim 30, wherein the CD123 antibodyis AbX₁.
 32. The method of claim 31, wherein AbX₁ comprises the heavychain variable region CDR sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQID NO: 3 and the light chain variable region CDR sequences SEQ ID NO: 4,SEQ ID NO: 5, and SEQ ID NO:
 6. 33. The method of claim 31, wherein AbX₁comprises a heavy chain variable region domain at least about 90%identical to SEQ ID NO: 7 and a light chain variable region at leastabout 90% identical to SEQ ID NO:
 9. 34. The method of claim 31, whereinAbX₁ comprises a heavy chain variable region domain at least about 90%identical to SEQ ID NO: 11 and a light chain variable region at leastabout 90% identical to SEQ ID NO:
 14. 35. The method of claim 31,wherein AbX₁ comprises a heavy chain variable region domain at leastabout 90% identical to SEQ ID NO: 12 and a light chain variable regionat least about 90% identical to SEQ ID NO:
 14. 36. The method of claim30, wherein the CD123 antibody is AbX₂.
 37. The method of claim 36,wherein AbX₂ comprises a heavy chain variable region domain at leastabout 90% identical to SEQ ID NO: 8 and a light chain variable region atleast about 90% identical to SEQ ID NO:
 10. 38. The method of claim 36,wherein AbX₂ comprises a heavy chain variable region domain at leastabout 90% identical to SEQ ID NO: 13 and a light chain variable regionat least about 90% identical to SEQ ID NO:
 15. 39. The method of any oneof claims 29-38, wherein the benzodiazepine is D5 or D5(a).